Systemic treatment of metastatic and/or systemically-disseminated cancers using GM-CSF-expressing poxviruses

ABSTRACT

The present invention concerns methods and compositions for the treatment of cancer and cancer cells using intravascular administration of a vaccinia virus. In some embodiments, methods and compositions involve a replicative vaccinia virus that encodes GM-CSF.

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 60/714,679 filed Sep. 7, 2005, which is incorporated herein byreference in its entirety.

BACKGROUND OF THE INVENTION

I. Field of the Invention

The present invention relates generally to the fields of oncology andvirology. More particularly, it concerns vaccinia viruses that expressGM-CSF and their use in systemic administration to treat cancer.

II. Description of Related Art

Normal tissue homeostasis is a highly regulated process of cellproliferation and cell death. An imbalance of either cell proliferationor cell death can develop into a cancerous state (Solyanik et al., 1995;Stokke et al., 1997; Mumby and Walter, 1991; Natoli et al., 1998;Magi-Galluzzi et al., 1998). For example, cervical, kidney, lung,pancreatic, colorectal and brain cancer are just a few examples of themany cancers that can result (Erlandsson, 1998; Kolmel, 1998; Mangrayand King, 1998; Gertig and Hunter, 1997; Mougin et al., 1998). In fact,the occurrence of cancer is so high that over 500,000 deaths per yearare attributed to cancer in the United States alone.

The maintenance of cell proliferation and cell death is at leastpartially regulated by proto-oncogenes and tumor suppressors. Aproto-oncogene or tumor suppressor can encode proteins that inducecellular proliferation (e.g., sis, erbB, src, ras and myc), proteinsthat inhibit cellular proliferation (e.g., Rb, p16, p19, p21, p53, NF1and WT1) or proteins that regulate programmed cell death (e.g., bcl-2)(Ochi et al., 1998; Johnson and Hamdy, 1998; Liebermann et al., 1998).However, genetic rearrangements or mutations of these proto-oncogenesand tumor suppressors result in the conversion of a proto-oncogene intoa potent cancer-causing oncogene or of a tumor suppressor into aninactive polypeptide. Often, a single point mutation is enough toachieve the transformation. For example, a point mutation in the p53tumor suppressor protein results in the complete loss of wild-type p53function (Vogelstein and Kinzler, 1992).

Currently, there are few effective options for the treatment of manycommon cancer types. The course of treatment for a given individualdepends on the diagnosis, the stage to which the disease has developedand factors such as age, sex and general health of the patient. The mostconventional options of cancer treatment are surgery, radiation therapyand chemotherapy. Surgery plays a central role in the diagnosis andtreatment of cancer. Typically, a surgical approach is required forbiopsy and to remove cancerous growth. However, if the cancer hasmetastasized and is widespread, surgery is unlikely to result in a cureand an alternate approach must be taken. Radiation therapy,chemotherapy, and immunotherapy are alternatives to surgical treatmentof cancer (Mayer, 1998; Ohara, 1998; Ho et al., 1998). Radiation therapyinvolves a precise aiming of high energy radiation to destroy cancercells and much like surgery, is mainly effective in the treatment ofnon-metastasized, localized cancer cells. Side effects of radiationtherapy include skin irritation, difficulty swallowing, dry mouth,nausea, diarrhea, hair loss and loss of energy (Curran, 1998; Brizel,1998).

Chemotherapy, the treatment of cancer with anti-cancer drugs, is anothermode of cancer therapy. The effectiveness of a given anti-cancer drugtherapy often is limited by the difficulty of achieving drug deliverythroughout solid tumors (el-Kareh and Secomb, 1997). Chemotherapeuticstrategies are based on tumor tissue growth, wherein the anti-cancerdrug is targeted to the rapidly dividing cancer cells. Most chemotherapyapproaches include the combination of more than one anti-cancer drug,which has proven to increase the response rate of a wide variety ofcancers (U.S. Pat. Nos. 5,824,348; 5,633,016 and 5,798,339, incorporatedherein by reference). A major side effect of chemotherapy drugs is thatthey also affect normal tissue cells, with the cells most likely to beaffected being those that divide rapidly in some cases (e.g., bonemarrow, gastrointestinal tract, reproductive system and hair follicles).Other toxic side effects of chemotherapy drugs can include sores in themouth, difficulty swallowing, dry mouth, nausea, diarrhea, vomiting,fatigue, bleeding, hair loss and infection.

Immunotherapy, a rapidly evolving area in cancer research, is yetanother option for the treatment of certain types of cancers.Theoretically, the immune system may be stimulated to identify tumorcells as being foreign and targets them for destruction. Unfortunately,the response typically is not sufficient to prevent most tumor growth.However, recently there has been a focus in the area of immunotherapy todevelop methods that augment or supplement the natural defense mechanismof the immune system. Examples of immunotherapies currently underinvestigation or in use are immune adjuvants (e.g., Mycobacterium bovis,Plasmodium falciparum, dinitrochlorobenzene and aromatic compounds)(U.S. Pat. Nos. 5,801,005 and 5,739,169; Hui and Hashimoto, 1998;Christodoulides et al., 1998), cytokine therapy (e.g., interferons(IL-1, GM-CSF and TNF) (Bukowski et al., 1998; Davidson et al., 1998;Hellstrand et al., 1998), and gene therapy (e.g., TNF, IL-1, IL-2, p53)(Qin et al., 1998; Austin-Ward and Villaseca, 1998; U.S. Pat. Nos.5,830,880 and 5,846,945) and monoclonal antibodies (e.g.,anti-ganglioside GM2, anti-HER-2, anti-p185) (Pietras et al., 1998;Hanibuchi et al., 1998; U.S. Pat. No. 5,824,311). Such methods, whileshowing some promise, have demonstrated limited success.

Replication-selective oncolytic viruses hold promise for the treatmentof cancer (Kim et al., 2001). These viruses can cause tumor cell deaththrough direct replication-dependent and/or viral geneexpression-dependent oncolytic effects (Kim et al., 2001). In addition,viruses are able to enhance the induction of cell-mediated antitumoralimmunity within the host (Todo et al., 2001; Sinkovics et al., 2000).These viruses also can be engineered to expressed therapeutic transgeneswithin the tumor to enhance antitumoral efficacy (Hermiston, 2000).

However, major limitations exist to this therapeutic approach. Althougha degree of natural tumor-selectivity can be demonstrated for some virusspecies, new approaches are still needed to engineer and/or enhancetumor-selectivity for oncolytic viruses in order to maximize safety.This selectivity will become particularly important when intravenousadministration is used, and when potentially toxic therapeutic genes areadded to these viruses to enhance antitumoral potency; gene expressionwill need to be tightly limited in normal tissues. In addition,increased antitumoral potency through additional mechanisms such asinduction of antitumoral immunity or targeting of the tumor-associatedvasculature is highly desirable.

Therefore, more effective and less toxic therapies for the treatment ofcancer are needed. The use of oncolytic viruses and immunotherapypresent areas that can be developed, however, the limitations discussedabove need to be overcome. Thus, the present invention addresses thoselimitations.

SUMMARY OF THE INVENTION

Thus, in accordance with the present invention there is provided amethod of killing a cancer cell in a subject comprising administering tothe subject an effective amount of a replicative vaccinia virus havingan expression region with a promoter directing expression of a nucleicacid encoding granulocyte-macrophage colony stimulating factor (GM-CSF),wherein the administration is intravascular. It is specificallycontemplated that the nucleic acid encodes human GM-CSF.

The vaccinia virus may be administered intravenously or intraarterially,for example, using intravenous drip or bolus, or using a pump. Thevaccinia virus may be dispersed in a pharmaceutically acceptableformulation. The subject may be administered between about 10⁵, 10⁶ 10⁷,10⁸ and about 10⁹, 10¹⁰, 10¹², 10¹³ pfu of virus, or between about 10⁷and about 10¹⁰ pfu of virus. The subject may be administered thevaccinia virus multiple times (1, 2, 3, 4, 5, 6, or more times), forexample, wherein the second treatment occurs within 1, 2, 3, 4, 5, 6, 7days or weeks of a first treatment, or wherein the second treatmentoccurs within 2 weeks of the first treatment. The same or a differentdose may be administered. The cancer cell may be a metastasized cancercell. The subject may have brain cancer, head & neck cancer, renalcancer, ovarian cancer, testicular cancer, uterine cancer, stomachcancer, lung cancer, colorectal cancer, breast cancer, prostate cancer,pancreatic cancer, hepatocellular cancer, leukemia, lymphoma, myeloma,or melanoma.

The vaccinia virus may have a deletion in its genome or a mutation inone or more genes. The thymidine kinase gene of the vaccinia virus mayhave been deleted. The vaccinia virus may have a mutation in a geneencoding (a) vaccinia virus growth factor; (b) a functionalinterferon-modulating polypeptide, wherein the interferon-modulatingpolypeptide directly binds interferon; (c) a complement controlpolypeptide, wherein the mutation results in the virus lacking at leastone functional complement control polypeptide; (d) a TNF-modulatingpolypeptide, wherein the mutation results in the virus lacking at leastone functional TNF-modulating polypeptide; (e) a serine proteaseinhibitor, wherein the mutation results in the virus lacking at leastone functional serine protease inhibitor; (f) an IL-1β modulatorpolypeptide, wherein the mutation results in the virus lacking at leastone functional IL-1β modulator polypeptide; (g) a functional A41L, B7R,N1L or vCKBP chemokine binding polypeptide or C11R EGF-like polypeptide,wherein the mutation results in the virus lacking at least one functionof A41L, B7R, N1L, vCKBP, or C11R; or (h) a polypeptide, wherein themutation results in an increase in infectious EEV foam of vacciniavirus. In addition, the vaccinia virus may comprise a mutation in avaccinia virus growth factor. The vaccinia virus may be the Wyeth orWestern Reserve (WR) strain. The promoter may be a vaccinia viruspromoter, a synthetic promoter, a promoter that directs transcriptionduring at least the early phase of infection, or a promoter that directstranscription during at least the late phase of infection.

In another embodiment, there is provided a method for treating cancer ina subject comprising administering to the subject an effective amount ofa replicative vaccinia virus having an expression region with a promoterdirecting expression of a nucleic acid encoding granulocyte-macrophagecolony stimulating factor (GM-CSF), wherein the administration isintravascular.

In yet another embodiment, there is provided a method for treating oneor more metastases in a subject comprising administering to the subjectan effective amount of a replicative vaccinia having an expressionregion with a promoter directing expression of a nucleic acid encodinggranulocyte-macrophage colony stimulating factor (GM-CSF), wherein theadministration is intravascular.

In other embodiments it is contemplated that methods involving areplication-competent vaccinia virus that is administeredintravascularly may contain a nucleic acid encoding a protein or RNAother than GM-CSF. In particular embodiments, the nucleic acid encodesanother cytokine. In certain embodiments, the nucleic acid encodes otherimmunostimulatory cytokines or chemokines, such as IL-12, IL-2 andothers. In additional embodiments, the nucleic acid may encode thymidinedeaminase or tumor necrosis factor (TNF), such as TNF-α. Moreover, it iscontemplated that replicative vaccinia viruses may express more than oneheterologous sequence. It may express, for example, GM-CSF protein andanother protein or RNA molecule.

It is specifically contemplated that any embodiment discussed withrespect to a particular method or composition may be implemented withrespect to other methods and compositions of the invention.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1—JX-594 intravenous (IV) treatment of spontaneous rathepatocellular carcinoma (HCC). Rats received the mutagenN-Nitrosomorpholine (NMM) in their drinking water (175 mg/L) for aperiod of 8 weeks and were then followed by ultrasound (US) until HCCtumors had formed and were 300-400 mm³ (typically after 16-20 weeks).Animals then received 3 intravenous doses (one every two weeks, arrows)of either PBS (n=17) or 1×10⁸ PFU of JX-594 virus (n=6). Subsequenttumor volumes were then calculated based on tumor measurements from USimaging.

FIGS. 2A-2B—JX-594 intravenous dose treatment of VX2 liver tumors inrabbits, efficacy against primary tumor and metastases. VX2 cells (froma dissociated 1 mm³ tumor) were implanted into the liver of New Zealandwhite rabbits and tumor growth followed by ultrasound (US) and CT scan.Once tumors reached 2-4 cm³ animals were treated with a single dose ofPBS (n=7) or JX-594 (1×10⁹ PFU), via intravenous or US guided ITinjection (n=3/group). (FIG. 2A) Subsequent tumor volume in the liverwas measured 7 weeks later by CT scan and (FIG. 2B) number of detectabletumor metastases in the lungs were counted following CT scan at weeks 6and 7.

FIG. 3—JX-594 and JX-963 lower dose intravenous repeat treatments of VX2liver tumors in rabbits. Tumor cells were implanted as described in(FIGS. 2A-2B). Animals were treated intravenously 3 times (every twoweeks, arrows) after tumors reached 2-4 cm³ with 1×10⁸ PFU of JX-594,JX-963 or vvDD (n=6/group), or PBS (n=18). Subsequent primary tumorvolume was followed by CT scan.

FIG. 4—Effects of JX-594, vvDD and JX-963 on lung metastases in rabbitsbearing VX2 liver tumors. Animals (from studies described in FIG. 3)were examined for liver metastases by CT scan at weekly intervals afterthe beginning of therapy. The mean number of detectable metastases peranimal in each group is shown.

FIG. 5—Survival of rabbits bearing VX2 liver tumors after IV delivery ofJX-963. Animals bearing liver tumors were treated with 3 doses of 1×10⁸PFU of JX-963 as described in FIG. 3. A Kaplin-Meier survival curve ofthese animals and the control treated group are shown. As the JX-594 andvvDD groups did not show significant differences in survival, JX594 andvvDD groups were not included.

FIGS. 6A-6C—Burst ratio of vaccinia strains, cytopathic effect, andsystemic delivery of viral strains to tumors. (FIG. 6A) Burst ratio ofvaccinia strains in tumor to normal cells. Different vaccinia strainswere used to infect both primary normal cells (NHBE) and a tumor cellline (A2780) at a Multiplicity of infection (MOI) of 1.0 Plaque FormingUnit (PFU)/cell. Virus collected 48 h later was titered by plaque assayand the ratio of virus produced (per cell) in tumor to normal cells isrepresented. (FIG. 6B) Cytopathic effect produced by viral infection.Western Reserve, Adenovirus serotype 5 and Adenovirus strain dl1520(ONYX-015) (in some assays) were added to cell lines at ranges of MOIs(PFU/cell), and cell viability measured after 72 hours using MTS(Promega). The MOI of virus (PFU/cell) needed to reduce the cellviability to 50% of untreated control wells (ED₅₀) is plotted. (FIG. 6C)Systemic delivery of viral strains to tumors. 1×10⁹ PFU of vacciniastrain Western Reserve or Adenovirus serotype 5 were deliveredintravenously to immunocompetent mice bearing subcutaneous CMT 64 or JCtumors. Mice were sacrificed after 48 or 72 hours andimmunohistochemistry performed against viral coat proteins on paraffinembedded sections of tumor tissue. Graphs show scoring of positive cellsin each tumour (*=none detectable). For each condition results are basedon tumours from 3 mice, and for each tumour, ten randomly chosen fieldsof view were scored.

FIG. 7—Cytopathic effect of WR and vvDD on a panel of human tumor celllines. EC₅₀ values were determined 72 h following infection of tumorcell lines with WR or vvDD. The MOI of virus (PFU/cell) needed to reducethe cell viability to 50% of untreated control wells (ED50) is plotted

FIGS. 8A-8C—Effects of overexpression of H-Ras on viral replication,biodistribution of WR and vvDD following systemic delivery to tumorbearing mice, and viral gene expression quantified by light production.(FIG. 7A) Effects of overexpression of H-Ras on viral replication. NIH3T3 cells, and NIH 3T3 cells expressing activated H-Ras, eitherproliferating or serum starved, were infected with different strains ofvaccinia at an MOI of 1.0 PFU/cell. Viral strains were parental WesternReserve (WR), and WR containing deletions or insertions in either theThymidine Kinase (TK) gene (vJS6), the viral growth factor (VGF) gene(vSC20), or containing deletions in both these genes (vvDD). Infectiousvirus was titered by plaque assay after 48 h. (FIG. 7B). Biodistributionof WR and vvDD following systemic delivery to tumor bearing mice.Athymic CD1 nu/nu mice bearing subcutaneous human HCT 116 tumors(arrowed) were treated with 1×10⁷ PFU of vaccinia strains via tail veininjection. Viral strains (WR and vvDD) expressed luciferase, and thesubsequent biodistribution of viral gene expression was detected bybioluminescence imaging in an IVIS 100 system (Xenogen Corp, Alameda)following addition of the substrate luciferin at the times indicatedafter treatment. (FIG. 7C) Viral gene expression, as quantified by lightproduction, was plotted over time for the regions of interest coveringthe whole body (ventral image)(dashed line, open symbols), or from thetumor only (dorsal view)(solid line, filled symbols) for BALB/c micebearing subcutaneous JC tumors (n=5 mice/group) and treated with 1×10⁷PFU of either virus by tail vein injection.

FIGS. 9A-9C—Rabbits bearing VX2 tumors implanted into the liver werefollowed by CT imaging at times after tumor implantation, CTL assayagainst VX2 tumor cells, and rabbits re-treated with JX-963. (FIG. 9A)Rabbits bearing VX2 tumors implanted into the liver were followed by CTimaging at times after tumor implantation. 1×10⁹ PFU of viruses vvDD andJX-963 were delivered by ear vein injection at 2, 3 and 4 weeks aftertumor implantation (arrows), when tumors measured 5 cm³. The number ofdetectable lung metastases was also measured in these animals(representative CT images of primary liver tumors are shown at 8 weeks)(n=18 for control treated animals; n=6 for vvDD treated; n=6 for JX-963treated). (FIG. 9B) CTL assay targeting VX2 tumor cells. CTL assay wasperformed by FACS analysis using pre-labeled VX2 cells mixed with 12.5×;25× and 50× unlabelled peripheral blood lymphocytes from rabbits bearingVX2 tumors and treated with JX-963; from untreated animals with VX2tumors; and from naïve animals. Cell death was quantified by the ACT1assay (Cell Technology, Mountain View). (FIG. 9C) Four Rabbits treatedas in (A) with JX-963 were re-treated with 1×10⁹ PFU of JX-963 at Day 42after implantation (arrow), subsequent tumor volume was followed by CTscan.

FIGS. 10A-10B—Viral production of cell lines infected with either WR orAd5, and cytopathic effect produced by viral infection. (FIG. 10A)Different cell lines were infected with either Western Reserve orAdenovirus serotype 5 at an MOI of 1.0 PFU/cell. Amounts of virusproduced (Infectious Units/cell) 48 h later were titered by plaqueassay. (FIG. 10B) Mice treated as in FIG. 1C were sacrificed and tumorsections stained for viral coat proteins. Representative photographsshow sections at 72 h and 10 days post-treatment.

FIG. 11—Viral production of cell lines infected with either WR or Ad5.Human tumor cell lines (Panc-1 and MCF-7) or human immortalized butnon-transformed cell lines (Beas-2B and MRC-5), either proliferating orgrown to contact inhibition (N.B. tumor cells did not become contactinhibited), were treated with different strains of vaccinia at an MOI of1.0 PFU/cell. Strains used were Western Reserve (WR) and WR containingdeletions in either the Thymidine Kinase (TK) gene (vJS6), the viralgrowth factor (VGF) gene (vSC20), or containing deletions in both thesegenes (vvDD). Virus produced after 48 h was titered by plaque assay.

FIG. 12—Recovery of systemically delivered vvDD. Recovery of vvDDdelivered systemically (intraperitoneal injection of 1×10⁹ PFU) toC57B/6 mice bearing subcutaneous MC38 tumors. Mice were sacrificed ondays 5 or 8 after treatment (n=8/group) and different tissues recoveredand viral infectious units (PFU/mg tissue) titered by plaque assay(*=below the limits of detection).

FIGS. 13A-13B—Efficacy of vvDD following delivery by different routesinto tumor bearing mouse models. (FIG. 13A) Single intravenousinjections of 1×10⁹ PFU of viral strain vvDD or vaccinia Wyeth strainbearing a Thymidine Kinase deletion were delivered to immunocompetentmice bearing subcutaneous TIB 75 tumors (50-100 mm³). Tumor volume wasmeasured by calipers, (n=8/group). (FIG. 13B) 1×10⁹ PFU of vvDD wasdelivered intratumorally (IT) or intraperitoneally (IP) to either SCIDmice bearing subcutaneous HT29 tumors or C57B/6 mice bearingsubcutaneous MC38 tumors and subsequent tumor volume compared to anuninfected control group (n=8/group).

FIG. 14—Formation of neutralizing antibodies following treatment of VX2tumor bearing rabbits with JX-963 (1×10⁸ PFU). Dilutions of plasmaobtained from rabbits at indicated times were incubated with a knownnumber of viral PFU, and dilutions required to retain 50% of the plaquesare shown (n=3).

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

GM-CSF-expressing poxviruses demonstrated efficacy and safety followingintratumoral injection in animals and in patients with melanoma(Mastrangelo et al., 1999; 2000). Localized injection site and distanteffects were seen, including tumor regressions and stabilizations(Mastrangelo et al., 1999). Efficacy was proposed to be due to inductionof an immune response to the cancer cells in the mammal. U.S. Pat. Nos.6,093,700 and 6,475,999 propose the use of poxviruses to deliver GM-CSFto tumors by intratumoral injection in humans in order to immunize insitu against melanoma, head and neck cancer, prostate cancer and bladder(Mastrangelo et al., 2002). These cancers were selected because of theirsuperficial nature and access to direct intratumoral injection. Thesecancers are also reportedly sensitive to immunotherapy approaches thatinduce systemic tumor-specific, cytotoxic T-lymphocytes. GM-CSF wasdemonstrated to be a potent inducer of tumor-specific cytotoxicT-lymphocytes (CTLs) (Dranoff et al., 1993). Since viruses induce immunecell infiltration and proinflammatory cytokines, viruses may constitutean ideal method of delivering and expressing GM-CSF within a tumor mass.

The intratumoral route of injection has major limitations, however. Thislimits treatment to tumors that are accessible to safe intratumoralinjection, usually superficial tumors, such as those listed above. Inaddition, efficacy against non-injected tumors requires induction ofsufficiently potent tumor-specific, cytotoxic T-lymphocytes. Theseweaknesses limit the utility of this approach to cancers that aresuperficial and are able to induce sufficiently potent tumor-specific,cytotoxic T-lymphocytes systemically. Such tumors are rare. The onlyclear example of applicability to this approach is metastatic melanoma.Even in this tumor type however, distant responses were slow and werelimited to superficial skin metastases. Organ-based or visceral tumorsdid not respond. Since these tumors are the cause of almost allcancer-related morbidity and deaths, systemic cytotoxic T-lymphocytes donot appear to be sufficient to induce lasting systemic, visceralresponses or cures. Therefore, the vast majority of human tumors are notamenable to successful efficacy with this approach.

Intravascular administration (i.e., intravenous, intra-arterial) withdelivery to metastatic tumors and immune tissues (e.g.,reticuloendothelial cells) has numerous theoretical advantages. First,delivery of GM-CSF-expressing poxviruses to the majority of tumor sites,or all tumor sites, in the body of the mammal allows for systemic tumordestruction by both local intratumoral effects (due to poxvirusreplication and GM-CSF effects locally within the infected tumors) andtumor-specific, cytotoxic T-lymphocytes induction and subsequentefficacy both at the infected tumor site and at a distance (in tumorsites that were not directly infected with the initial dosage). Deliveryof virus to tumors through the bloodstream also allows for more uniform,widespread infection of cancer cells in the tumor. Therefore, theinventors now propose that multi-focal, disseminated, metastatic tumorscan be effectively treated by intravascular poxviruses expressingGM-CSF. Many cancer types and/or stages that would not be amenable tothe intratumoral approach would also be potentially treatable with theintravascular injection approach. Examples would include, but would notbe limited to, lung, colorectal, breast, prostate, pancreatic,hepatocellular, leukemias, lymphomas, myelomas, and melanomas.

However, the safe and effective use of intravascular poxvirusesexpressing GM-CSF was viewed by the skilled artisan as being negativelyimpacted by potential problems. First, safety concerns were significant.After intravascular administration, numerous normal tissues would beexposed to poxvirus infection. Subsequent expression of a potentproinflammatory cytokine like GM-CSF would be predicted to potentiallylead to significant inflammation in numerous organs such as liver, lung,kidney, heart, brain and others. In addition, systemic exposure toviremia can potentially induce sepsis and its associated complications(e.g., hypotension). Poxvirus infection in the brain of mice or humans,for example, can lead to clinically-significant, even fatal,encephalitis. GM-CSF expression could potentially significantly worsenthis complication due to enhancement of inflammation.

Second, systemic induction of tumor-specific CTLs through GM-CSFexpression was previously performed only through localized GM-CSFexpression either through direct intratumoral injection (e.g.,Vaccinia-GM-CSF, HSV-GM-CSF) or through ex vivo infection/transfectionof autologous or allogeneic tumor cells to express GM-CSF (e.g., GVAXapproach) followed by injection of GM-CSF cells into the skin.

Despite these potential drawbacks, the inventors now have demonstratedthat two different GM-CSF-expressing poxviruses were well-toleratedintravenously and highly effective against disseminated tumors andmetastases. In addition, a GM-CSF-expressing poxvirus had significantlybetter efficacy against both primary tumors and lung metastases than itsnon-GM-CSF-expressing control after intravenous administration. Also,this virus had significantly better efficacy against both primary tumorsand lung metastases than a comparable virus (Wyeth vaccine strain)despite an additional deletion in the vgf gene not present in the othervirus. Therefore, intravenous administration with a vaccinia expressinghuman GM-CSF resulted in significantly better efficacy over the samevaccinia without GM-CSF, and intravascular administration of a WR straindeletion mutant expressing human GM-CSF was significantly better than astandard vaccine strain expressing GM-CSF. These viruses werewell-tolerated after intravenous administration to both tumor-bearing(rats and rabbits) and normal animals (rabbits). Treated animals did notlose weigh while tumor-bearing animals that did not receive treatmentlost weight. Survival was increased following intravenous treatment. Nosignificant organ toxicity was noted by blood testing or histopathology.The only reproducible histopathological findings were multiple sites oflymphoid hyperplasia that were noted following treatment (consistentwith systemic immunostimulation). No significant toxicities were notedon histopathology. Therefore, these GM-CSF expressing poxviruses arewell-tolerated at doses that were highly effective against systemiccancer.

I. POXVIRUSES

A. Vaccinia Virus

Vaccinia virus is a mystery to virology. It is not known whethervaccinia virus is the product of genetic recombination, if it is aspecies derived from cowpox virus or variola virus by prolonged serialpassage, or if it is the living representative of a now extinct virus.Vaccinia virus was used for smallpox vaccination via inoculation intothe superficial layers of the skin of the upper arm. However, with theeradication of smallpox, routine vaccination with vaccinia virus hasceased. Recent interest in vaccinia has focused on its possible usage asa vector for immunization against other viruses and gene therapy.

Vaccinia virus is a member of the family Poxviridae, the subfamilyChordopoxvirinae and the genus Orthopoxvirus. The virions contain RNApolymerase, early transcription factor, poly(A) polymerase, cappingenzyme complex, RNA (nucleoside-2′) methyltransferase, nucleosidetriphosphate phosphohydrolase II, nick-joining enzyme, DNA topoisomeraseand protein kinase. The genome is a double-stranded DNA of just over180,000 base pairs characterized by a 10 kB inverted terminal repeat.The virus enters cells through pH-independent fusion with the plasmamembrane or a low pH-dependent endosomal route.

B. Other Poxviruses

The genus Orthopoxvirus is relatively more homogeneous than othermembers of the Chordopoxvirinae subfamily and includes 11 distinct butclosely related species, which includes vaccinia virus, variola virus(causative agent of smallpox), cowpox virus, buffalopox virus, monkeypoxvirus, mousepox virus and horsepox virus species as well as others (seeMoss, 1996). Certain embodiments of the invention, as described herein,may be extended to other members of Orthopoxvirus genus as well as theParapoxvirus, Avipoxvirus, Capripoxvirus, Leporipoxvirus, Suipoxvirus,Molluscipoxvirus, and Yatapoxvirus genus. A genus of poxvirus family isgenerally defined by serological means including neutralization andcross-reactivity in laboratory animals. Various members of theOrthopoxvirus genus, as well as other members of the Chordovirinaesubfamily utilize immunomodulatory molecules, examples of which areprovided herein, to counteract the immune responses of a host organism.Thus, the invention described herein is not limited to vaccinia virus,but may be applicable to a number of viruses.

II. ENGINEERING OF POXVIRUSES

Viruses are frequently inactivated, inhibited or cleared byimmunomodulatory molecules such as interferons (-α, -β, -γ) and tumornecrosis factor-α (TNF) (Moss, 1996). Host tissues andinflammatory/immune cells frequently secrete these molecules in responseto viral infection. These molecules can have direct antiviral effectsand/or indirect effects through recruitment and/or activation ofinflammatory cells and lymphocytes. Given the importance of theseimmunologic clearance mechanisms, viruses have evolved to express geneproducts that inhibit the induction and/or function of thesecytokines/chemokines and interferons. For example, vaccinia virus (VV;and some other poxviruses) encodes the secreted protein vCKBP (B29R)that binds and inhibits the CC chemokines (e.g., RANTES, eotaxin,MIP-1-alpha) (Alcami et al., 1998). Some VV strains also express asecreted viral protein that binds and inactivates TNF (e.g., ListerA53R) (Alcami et al., 1999). Most poxvirus strains have genes encodingsecreted proteins that bind and inhibit the function of interferons-α/β(e.g., B18R) or interferon-γ (B8R). vC12L is an IL-18-binding proteinthat prevents IL-18 from inducing IFN-γ and NK cell/cytotoxic T-cellactivation.

Most poxvirus virulence research has been performed in mice. Many, butnot all, of these proteins are active in mice (B18R, for example, isnot). In situations in which these proteins are active against the mouseversions of the target cytokine, deletion of these genes leads toreduced virulence and increased safety with VV mutants with deletions ofor functional mutations in these genes. In addition, theinflammatory/immune response to and viral clearance of these mutants isoften increased compared to the parental virus strain that expresses theinhibitory protein. For example, deletion of the T1/35 kDa family ofpoxvirus-secreted proteins (chemokine-binding/-inhibitory proteins) canlead to a marked increase in leukocyte infiltration into virus-infectedtissues (Graham et al., 1997). Deletion of the vC12L gene in VV leads toreduced viral titers/toxicity following intranasal administration inmice; in addition, NK cell and cytotoxic T-lymphocyte activity isincreased together with IFN-γ induction (Smith et al., 2000). Deletionof the Myxoma virus T7 gene (able to bind IFN-γ and a broad range ofchemokines) results in reduced virulence and significantly increasedtissue inflammation/infiltration in a toxicity model (Upton et al.,1992; Mossman et al., 1996). Deletion of the M-T2 gene from myxoma virusalso resulted in reduced virulence in a rabbit model (Upton et al.1991). Deletion of the B18R anti-interferon-α/-β gene product also leadsto enhanced viral sensitivity to IFN-mediated clearance, reduced titersin normal tissues and reduced virulence (Symons et al., 1995; Colamoniciet al., 1995; Alcami et al., 2000). In summary, these viral geneproducts function to decrease the antiviral immune response andinflammatory cell infiltration into virus-infected tissues. Loss ofprotein function through deletion/mutation leads to decreased virulenceand/or increased proinflammatory properties of the virus within hosttissues. See PCT/US2003/025141, which is hereby incorporated byreference.

Cytokines and chemokines can have potent antitumoral effects (Vicari etal., 2002; Homey et al., 2002). These effects can be on tumor cellsthemselves directly (e.g., TNF) or they can be indirect through effectson non-cancerous cells. An example of the latter is TNF, which can haveantitumoral effects by causing toxicity to tumor-associated bloodvessels; this leads to a loss of blood flow to the tumor followed bytumor necrosis. In addition, chemokines can act to recruit (and in somecases activate) immune effector cells such as neutrophils, eosinophils,macrophages and/or lymphocytes. These immune effector cells can causetumor destruction by a number of mechanisms. These mechanisms includethe expression of antitumoral cytokines (e.g., TNF), expression offas-ligand, expression of perforin and granzyme, recruitment of naturalkiller cells, etc. The inflammatory response can eventually lead to theinduction of systemic tumor-specific immunity. Finally, many of thesecytokines (e.g., TNF) or chemokines can act synergistically withchemotherapy or radiation therapy to destroy tumors.

Clinically effective systemic administration of recombinant versions ofthese immunostimulatory proteins is not feasible due to (1) induction ofsevere toxicity with systemic administration and (2) local expressionwithin tumor tissue is needed to stimulate local infiltration andantitumoral effects. Approaches are needed to achieve high localconcentrations of these molecules within tumor masses while minimizinglevels in the systemic circulation. Viruses can be engineered to expresscytokine or chemokinc genes in an attempt to enhance their efficacy.Expression of these genes from replication-selective vectors haspotential advantages over expression from non-replicating vectors.Expression from replicating viruses can result in higher localconcentrations within tumor masses; in addition, replicating viruses canhelp to induce antitumoral immunity through tumor celldestruction/oncolysis and release of tumor antigens in a proinflammatoryenvironment. However, there are several limitations to this approach.Serious safety concerns arise from the potential for release into theenvironment of a replication-competent virus (albeit tumor-selective)with a gene that can be toxic if expressed in high local concentrations.Viruses that express potent proinflammatory genes from their genome maytherefore pose safety risks to the treated patient and to the generalpublic. Even with tumor-targeting, replication-selective virusesexpressing these genes, gene expression can occur in normal tissuesresulting in toxicity. In addition, size limitations prevent expressionof multiple and/or large genes from viruses such as adenovirus; thesemolecules will definitely act more efficaciously in combination.Finally, many of the oncolytic viruses in use express anti-inflammatoryproteins and therefore these viruses will counteract the induction of aproinflammatory milieu within the infected tumor mass. The result willbe to inhibit induction of antitumoral immunity, antivascular effectsand chemotherapy-/radiotherapy-sensitization.

A. Vaccinia Virus Products

1. Interferon-Modulating Polypeptides

Interferon-α/-β blocks viral replication through several mechanisms.Interferon-γ has weaker direct viral inhibitory effects but is a potentinducer of cell-mediated immunity through several mechanisms. Viruseshave evolved to express secreted gene products that are able tocounteract the antiviral effects of interferons. For example, vacciniavirus (and other poxviruses) encodes the secreted proteins B8R and B18Rwhich bind interferon-γ and -α/-β, respectively (Smith et al., 1997;Symons et al., 1995; Alcami et al., 2000). An additional example of avaccinia gene product that reduces interferon induction is the caspase-1inhibitor B13R which inhibits activation of the interferon-γ-inducingfactor IL-18. Interferon modulating polypeptides include, but are notlimited to, B18R, which may be termed B19R in other viral strains, suchas the Copenhagen strain of Vaccinia virus; B8R; B13R; vC12L; A53R; E3Land other viral polypeptides with similar activities or properties. IFNmodulating polypeptides may be divided into the non-exclusive categoriesof those that preferentially modulate IFNα and/or β pathways (such asB18R, B8R, B13R, or vC12L) and those that modulate IFNγ pathways (forexample B8R, B13R, or vC12L).

Cancer cells are frequently resistant to the effects of interferons. Anumber of mechanisms are involved. These include the fact that rassignal transduction pathway activation (e.g., by ras mutation, upstreamgrowth factor receptor overexpression/mutation, etc.), a common featureof cancer cells, leads to PKR inhibition. In addition, lymphocytes areoften inhibited in tumor masses by a variety of mechanisms includingIL-10 production and fas-L expression by tumor cells. Since lymphocytesare a major source of interferon-γ production, lymphocyte inhibitionleads to a decrease in interferon-γ production in tumors. Therefore,tumor masses tend to be sanctuaries from the effects of interferons. Inaddition, interferons themselves can have antitumoral effects. Forexample, IFN-γ can increase MHC class-I-associated antigen presentation;this will allow more efficient CTL-mediated killing of tumor cells.IFN-α/β, for example, can block angiogenesis within tumor masses andthereby block tumor growth.

2. Complement Control Polypeptides

A major mechanism for the clearance of viral pathogens is the killing ofinfected cells within the host or of virions within an organism bycomplement-dependent mechanisms. As the infected cell dies it is unableto continue to produce infectious virus. In addition, during apoptosisintracellular enzymes are released which degrade DNA. These enzymes canlead to viral DNA degradation and virus inactivation. Apoptosis can beinduced by numerous mechanisms including the binding of activatedcomplement and the complement membrane attack complex. Poxviruses suchas vaccinia have evolved to express gene products that are able tocounteract the complement-mediated clearance of virus and/orvirus-infected cells. These genes thereby prevent apoptosis and inhibitviral clearance by complement-dependent mechanisms, thus allowing theviral infection to proceed and viral virulence to be increased. Forexample, vaccinia virus complement control proteins (VCP; e.g., C21L)have roles in the prevention of complement-mediated cell killing and/orvirus inactivation (Isaacs et al., 1992). VCP also has anti-inflammatoryeffects since its expression decreases leukocyte infiltration intovirally-infected tissues. Complement control polypeptides include, butare not limited to, VCP, also known as C3L or C21L.

Cancer cells frequently overexpress cellular anti-complement proteins;this allows cancer cells to survive complement attack+/−tumor-specificantibodies (Caragine et al., 2002; Durrant et al., 2001; Andoh et al.2002). Therefore, agents that preferentially target tumor cells due totheir inherent resistance to complement-mediated killing would haveselectivity and potential efficacy in a wide range of human cancers(Durrant et al., 2001). In addition, one of the hallmarks of cancercells is a loss of normal apoptotic mechanisms (Gross et al., 1999).Resistance to apoptosis promotes carcinogenesis as well as resistance toantitumoral agents including immunologic, chemotherapeutic andradiotherapeutic agents (Eliopoulos et al., 1995). Apoptosis inhibitioncan be mediated by a loss of pro-apoptotic molecule function (e.g.,bax), an increase in the levels/function of anti-apoptotic molecules(e.g., bcl-2) and finally a loss of complement sensitivity.

3. TNF-Modulating Polypeptides

One of the various mechanisms for the clearance of viral pathogens isthe killing of infected cells within the host by the induction ofapoptosis, as described above. Apoptosis can be induced by numerousmechanisms including the binding of TNF and lymphotoxin-alpha (LTα) tocellular TNF receptors, which triggers intracellular signaling cascades.Activation of the TNF receptors function in the regulation of immune andinflammatory responses, as well as inducing apoptotic cell death(Wallach et al., 1999)

Various strains of poxviruses, including some vaccinia virus strains,have evolved to express gene products that are able to counteract theTNF-mediated clearance of virus and/or virus-infected cells. Theproteins encoded by these genes circumvent the proinflammatory andapoptosis inducing activities of TNF by binding and sequesteringextracellular TNF, resulting in the inhibition of viral clearance.Because viruses are not cleared, the viral infection is allowed toproceed, and thus, viral virulence is increased. Various members of thepoxvirus family express secreted viral TNF receptors (vTNFR). Forexample, several poxviruses encode vTNFRs, such as myxoma (T2 protein),cowpox and vaccinia virus strains, such as Lister, may encode one ormore of the CrmB, CrmC (A53R), CrmD, CrmE, B28R proteins and/orequivalents thereof. These vTNFRs have roles in the prevention ofTNF-mediated cell killing and/or virus inactivation (Saraiva and Alcami,2001). TNF modulatory polypeptides include, but are not limited to,A53R, B28R (this protein is present, but may be inactive in theCopenhagen strain of vaccinia virus) and other polypeptides with similaractivities or properties.

One of the hallmarks of cancer cells is aberrant gene expression, whichmay lead to a loss of sensitivity to a number of molecular mechanismsfor growth modulation, such as sensitivity to the anti-cancer activitiesof TNF. Thus, viral immunomodulatory mechanisms may not be required forthe propagation of a virus within the tumor microenvironment.

4. Serine Protease Inhibitors

A major mechanism for the clearance of viral pathogens is the inductionof apoptosis in infected cells within the host. As the infected celldies it is unable to continue to produce infectious virus. In addition,during apoptosis intracellular enzymes are released which degrade DNA.These enzymes can lead to viral DNA degradation and virus inactivation.Apoptosis can be induced by numerous mechanisms including the binding ofcytokines (e.g., tumor necrosis factor), granzyme production bycytotoxic T-lymphocytes or fas-ligand binding; caspase activation is acritical part of the final common apoptosis pathway. Viruses haveevolved to express gene products that are able to counteract theintracellular signaling cascade induced by such molecules includingfas-ligand or tumor necrosis factor (TNF)/TNF-related molecules (e.g.,E3 10.4/14.5, 14.7 genes of adenovirus (Wold et al., 1994); E1B-19 kD ofadenovirus (Boyd et al., 1994); crmA from cowpox virus; B13R fromvaccinia virus) (Dobbelstein et al., 1996; Kettle et al., 1997). Thesegene products prevent apoptosis by apoptosis-inducing molecules and thusallow viral replication to proceed despite the presence of antiviralapoptosis-inducing cytokines, fas, granzyme or other stimulators ofapoptosis.

VV SPI-2/B13R is highly homologous to cowpox CrmA; SPI-1 (VV) is weaklyhomologous to CrmA (Dobbelstein et al., 1996). These proteins areserpins (serine protease inhibitors) and both CrmA and SPI-2 have rolesin the prevention of various forms of apoptosis Inhibition ofinterleukin-1β-converting enzyme (ICE) and granzyme, for example, canprevent apoptosis of the infected cell. These gene products also haveanti-inflammatory effects. They are able to inhibit the activation ofIL-18 which in turn would decrease IL-18-mediated induction of IFN-γ.The immunostimulatory effects of IFN-γ on cell-mediated immunity arethereby inhibited (Kettle et al., 1997). SPIs include, but are notlimited to, B13R, B22R, and other polypeptides with similar activitiesor properties.

One of the hallmarks of cancer cells is a loss of normal apoptoticmechanisms (Gross et al., 1999). Resistance to apoptosis promotescarcinogenesis as well as resistance to antitumoral agents includingimmunologic, chemotherapeutic and radiotherapeutic agents (Eliopoulos etal., 1995). Apoptosis inhibition can be mediated by a loss ofpro-apoptotic molecule function (e.g., bax) or an increase in thelevels/function of anti-apoptotic molecules (e.g., bcl-2).

5. IL-1β-Modulating Polypeptides

IL-1β is a biologically active factors that acts locally and alsosystemically. Only a few functional differences between IL-1β and IL-1αhave been described. The numerous biological activities of IL-1β isexemplified by the many different acronyms under which IL-1 has beendescribed. IL-1 does not show species specificity with the exception ofhuman IL-1β that is inactive in porcine cells. Some of the biologicalactivities of IL-1 are mediated indirectly by the induction of thesynthesis of other mediators including ACTH (Corticotropin), PGE2(prostaglandin E2), PF4 (platelet factor-4), CSF (colony stimulatingfactors), IL-6, and IL-8. The synthesis of IL-1 may be induced by othercytokines including TNF-α, IFN-α, IFN-β and IFN-γ and also by bacterialendotoxins, viruses, mitogens, and antigens. The main biologicalactivity of IL-1 is the stimulation of T-helper cells, which are inducedto secrete IL-2 and to express IL-2 receptors. Virus-infectedmacrophages produce large amounts of an IL-1 inhibitor that may supportopportunistic infections and transformation of cells in patients withT-cell maturation defects. IL-1 acts directly on B-cells, promotingtheir proliferation and the synthesis of immunoglobulins. IL-1 alsofunctions as one of the priming factors that makes B-cells responsive toIL-5. IL-1 stimulates the proliferation and activation of NK-cells andfibroblasts, thymocytes, glioblastoma cells.

Blockade of the synthesis of IL-1β by the viral protein is regarded as aviral strategy allowing systemic antiviral reactions elicited by IL-1 tobe suppressed or diminished. Binding proteins effectively blocking thefunctions of IL-1 with similar activity as B15R have been found also tobe encoded by genes of the cowpox virus. Vaccinia virus also encodesanother protein, designated B8R, which behaves like a receptor forcytokines (Alcami and Smith, 1992; Spriggs et al., 1992). IL-1modulating polypeptides include, but are not limited to, B13R, B15R, andother polypeptides with similar activities or properties.

One of the hallmarks of cancer cells is aberrant gene expression, whichmay lead to a loss of sensitivity to a number of molecular mechanismsfor growth modulation, such as sensitivity to the anti-cancer activitiesof IL-1. Thus, viral immunomodulatory mechanisms may not be required forthe propagation of a virus within the tumor microenvironment.

6. EEV Form

Viral spread to metastatic tumor sites, and even spread within aninfected solid tumor mass, is generally inefficient (Heise et al.,1999). Intravenous administration typically results in viral clearanceor inactivation by antibodies (e.g., adenovirus) (Kay et al., 1997)and/or the complement system (e.g., HSV) (Ikeda et al., 1999). Inaddition to these immune-mediated mechanisms, the biodistribution ofthese viruses results in the vast majority of intravenous virusdepositing within normal tissues rather than in tumor masses.Intravenous adenovirus, for example, primarily ends up within the liverand spleen; less than 0.1% of the input virus depositing within tumors,even in immunodeficient mice (Heise et al., 1999). Therefore, althoughsome modest antitumoral efficacy can be demonstrated with extremely highrelative doses in immunodeficient mouse tumor models, intravenousdelivery is extremely inefficient and significantly limits efficacy.

Vaccinia virus has the ability to replicate within solid tumors andcause necrosis. In addition, thymidine kinase-deletion mutants caninfect tumor masses and ovarian tissue and express marker genespreferentially in mouse tumor model systems (Gnant et al., 1999).However, since these studies generally determined tumor targeting basedon marker gene expression after ≧5 days, it is unclear whether the viruspreferentially deposits in, expresses genes in or replicates intumor/ovary tissue (Puhlmann et al., 2000). Regardless of the mechanism,the antitumoral efficacy of this virus without additional transgenes wasnot statistically significant (Gnant et al., 1999). In contrast,intratumoral virus injection had significant antitumoral efficacy(McCart et al. 2000). Therefore, i.v. efficacy could be improved if i.v.delivery to the tumor were to be improved.

Vaccinia virus replicates in cells and produces both intracellular virus(IMV, intracellular mature virus; IEV, intracellular enveloped virus)and extracellular virus (EEV, extracellular enveloped virus; CEV,cell-associated extracellular virus) (Smith et al., 1998). IMVrepresents approximately 99% of virus yield following replication bywild-type vaccinia virus strains. This virus form is relatively stablein the environment, and therefore it is primarily responsible for spreadbetween individuals; in contrast, this virus does not spread efficientlywithin the infected host due to inefficient release from cells andsensitivity to complement and/or antibody neutralization. In contrast,EEV is released into the extracellular milieu and typically representsonly approximately 1% of the viral yield (Smith et al., 1998). EEV isresponsible for viral spread within the infected host and is relativelyeasily degraded outside of the host. Importantly, EEV has developedseveral mechanisms to inhibit its neutralization within the bloodstream.First, EEV is relatively resistant to complement (Vanderplasschen etal., 1998); this feature is due to the incorporation of host cellinhibitors of complement into its outer membrane coat plus secretion ofVaccinia virus complement control protein (VCP) into local extracellularenvironment. Second, EEV is relatively resistant to neutralizingantibody effects compared to IMV (Smith et al., 1997). EEV is alsoreleased at earlier time points following infection (e.g., 4-6 hours)than is IMV (which is only released during/after cell death), andtherefore spread of the EEV form is faster (Blasco et al., 1993).

Unfortunately, however, wild-type vaccinia strains make only very smallamounts of EEV, relatively. In addition, treatment with vaccinia virus(i.e., the input dose of virus) has been limited to intracellular virusforms to date. Standard vaccinia virus (VV) manufacturing andpurification procedures lead to EEV inactivation (Smith et al., 1998),and non-human cell lines are frequently used to manufacture the virus;EEV from non-human cells will not be protected from complement-mediatedclearance (complement inhibitory proteins acquired from the cell by EEVhave species restricted effects). Vaccinia virus efficacy has thereforebeen limited by the relative sensitivity of the IMV form toneutralization and by its inefficient spread within solid tumor masses;this spread is typically from cell to adjacent cell. IMV spread todistant tumor masses, either through the bloodstream or lymphatics, isalso inefficient.

Therefore, the rare EEV form of vaccinia virus has naturally acquiredfeatures that make it superior to the vaccinia virus form used inpatients to date (IMV); EEV is optimized for rapid and efficient spreadthrough solid tumors locally and to regional or distant tumor sites.Since EEV is relatively resistant to complement effects, when it isgrown in a cell type from the same species, this virus form will haveenhanced stability and retain activity longer in the blood followingintravascular administration than standard preparations of vacciniavirus (which contain exclusively IMV) (Smith et al., 1998). Since EEV isresistant to antibody-mediated neutralization, this virus form willretain activity longer in the blood following intravascularadministration than standard preparations of vaccinia virus (whichcontain almost exclusively IMV) (Vanderplasschen et al., 1998). Thisfeature will be particularly important for repeat administration onceneutralizing antibody levels have increased; all approved anti-cancertherapies require repeat administration. Therefore, the EEV form ofvaccinia, and other poxviruses, will result in superior delivery oftherapeutic viruses and their genetic payload to tumors through thebloodstream. This will lead to enhanced systemic efficacy compared withstandard poxvirus preparations. Finally, the risk of transmission toindividuals in the general public should be reduced significantly sinceEEV is extremely unstable outside of the body. Polypeptides involved inthe modulation of the EEV form of a virus include, but are not limitedto, A34R, B5R, and various other proteins that influence the productionof the EEV form of the poxviruses. A mutation at codon 151 of A34R froma lysine to a aspartic acid (K151D mutation) renders the A34R proteinless able to tether the EEV form to the cell membrane. B5R is anEEV-membrane bound polypeptide that may bind complement. The totaldeletion of A43R may lead to increased EEV release, but markedly reducedinfectivity of the viruses, while the K151D mutation increases EEVrelease while maintaining infectivity of the released viruses. B5R hassequence homology to VCP (anti-complement), but complement inhibitionhas not yet been proven.

Briefly, one method for identifying a fortified EEV form is as follows.EEV are diluted in ice-cold MEM and mixed (1:1 volume) with active orheat-inactivated (56° C., 30 min, control) serum diluted in ice-cold MEM(final dilution of serum 1/10, 1/20, or 1/30). After incubation or 75minutes at 7° C., samples are cooled on ice and mAb 5B4/2F2 is added tofresh EEV samples to neutralize any contaminates (IMV and ruptured EEV).Virions are then bound to RK13 cells for one hour on ice, complement andunbound virions are washed away, and the number of plaques are countedtwo days later. The higher the plaque number the greater the resistanceto complement. Vanderplasschen et al. (1998), herein incorporated byreference. Exemplary methods describing the isolation of EEV forms ofVaccinia virus can be found in Blasco et al. (1992) (incorporated hereinby reference).

7. Other Polypeptides

Other viral immunomodulatory polypeptides may include polypeptides thatbind other mediators of the immune response and/or modulate molecularpathways associated with the immune response. For example, chemokinebinding polypeptides such as B29R (this protein is present, but may beinactive in the Copenhagen strain of Vaccinia virus), C23L, vCKBP, A41Land polypeptides with similar activities or properties. Other vacciniavirus proteins such as the vaccinia virus growth factor (e.g., C11L),which is a viral EGF-like growth factor, may also be the target foralteration in some embodiments of the invention. Other polypeptides thatmay be classified as viral immunomodulatory factors include, but are notlimited to B7R, N1L, or other polypeptides that whose activities orproperties increase the virulence of a poxvirus.

8. Vaccinia Virus-Induced Cell Fusion

In certain embodiments of the invention an alteration, deletion, ormutation of A56R or K2L encoding nucleic genes may lead to cell-cellfusion or syncitia formation induced by VV infection. Vacciniavirus-induced cell fusion will typically increase antitumoral efficacyof VV due to intratumoral viral spread. Intratumoral viral spreading bycell fusion will typically allow the virus to avoid neutralizingantibodies and immune responses. Killing and infection of adjacentuninfected cells (i.e., a “bystander” effect) may be more efficient inVV with mutations in one or both of these genes, which may result inimproved local antitumoral effects.

B. Virus Propagation

Vaccinia virus may be propagated using the methods described by Earl andMoss in Ausbel et al., Current Protocols in Molecular Biology, pages16.15.1 to 16.18.10, which is incorporated by reference herein.

III. PROTEINACEOUS AND NUCLEIC ACID COMPOSITIONS

The present invention concerns poxviruses that are advantageous in thestudy and treatment of cancer cells and cancer in a patient. It concernsvaccinia viruses, optionally constructed with one or more mutationscompared to wild-type such that the virus has desirable properties foruse against cancer cells, while being less toxic or non-toxic tonon-cancer cells. Such poxviruses are described in PCT/US2003/025141,which is incorporated herein by reference. The teachings described belowprovide various protocols, by way of example, of implementing methodsand compositions of the invention. They provide background forgenerating mutated viruses through the use of recombinant DNAtechnology.

A. Proteinaceous Compositions

In certain embodiments, the present invention concerns generatingvaccinia virus, optionally those that lack one or more functionalpolypeptides or proteins and/or generating poxviruses that have theability to release more of a particular form of the virus, such as aninfectious EEV form. In other embodiments, the present inventionconcerns poxviruses and their use in combination with proteinaceouscomposition as part of a pharmaceutically acceptable formulation.

As used herein, a “protein” or “polypeptide” refers to a moleculecomprising at least one amino acid residue. In some embodiments, awild-type version of a protein or polypeptide are employed, however, inmany embodiments of the invention, a viral protein or polypeptide isabsent or altered so as to render the virus more useful for thetreatment of a cancer cells or cancer in a patient. The terms describedabove may be used interchangeably herein. A “modified protein” or“modified polypeptide” refers to a protein or polypeptide whose chemicalstructure is altered with respect to the wild-type protein orpolypeptide. In some embodiments, a modified protein or polypeptide hasat least one modified activity or function (recognizing that proteins orpolypeptides may have multiple activities or functions). The modifiedactivity or function may we reduced, diminished, eliminated, enhanced,improved, or altered in some other way (such as specificity) withrespect to that activity or function in a wild-type protein orpolypeptide. It is specifically contemplated that a modified protein orpolypeptide may be altered with respect to one activity or function yetretain wild-type activity or function in other respects. Alternatively,a modified protein may be completely nonfunctional or its cognatenucleic acid sequence may have been altered so that the polypeptide isno longer expressed at all, is truncated, or expresses a different aminoacid sequence as a result of a frameshift.

In certain embodiments the size of a mutated protein or polypeptide maycomprise, but is not limited to, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140,150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325,350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675,700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1100,1200, 1300, 1400, 1500, 1750, 2000, 2250, 2500 or greater amino moleculeresidues, and any range derivable therein. It is contemplated thatpolypeptides may be mutated by truncation, rendering them shorter thantheir corresponding wild-type form.

As used herein, an “amino molecule” refers to any amino acid, amino acidderivative or amino acid mimic as would be known to one of ordinaryskill in the art. In certain embodiments, the residues of theproteinaceous molecule are sequential, without any non-amino moleculeinterrupting the sequence of amino molecule residues. In otherembodiments, the sequence may comprise one or more non-amino moleculemoieties. In particular embodiments, the sequence of residues of theproteinaceous molecule may be interrupted by one or more non-aminomolecule moieties.

Accordingly, the term “proteinaceous composition” encompasses aminomolecule sequences comprising at least one of the 20 common amino acidsin naturally synthesized proteins, or at least one modified or unusualamino acid.

Proteinaceous compositions may be made by any technique known to thoseof skill in the art, including the expression of proteins, polypeptidesor peptides through standard molecular biological techniques, theisolation of proteinaceous compounds from natural sources, or thechemical synthesis of proteinaceous materials. The nucleotide andprotein, polypeptide and peptide sequences for various genes have beenpreviously disclosed, and may be found at computerized databases knownto those of ordinary skill in the art. One such database is the NationalCenter for Biotechnology Information's GenBank and GenPept databases(www.ncbi.nlm.nih.gov). The coding regions for these known genes may beamplified and/or expressed using the techniques disclosed herein or aswould be know to those of ordinary skill in the art.

1. Functional Aspects

When the present application refers to the function or activity of viralproteins or polypeptides, it is meant to refer to the activity orfunction of that viral protein or polypeptide under physiologicalconditions, unless otherwise specified. For example, aninterferon-modulating polypeptide refers to a polypeptide that affectsat least one interferon and its activity, either directly or indirectly.The polypeptide may induce, enhance, raise, increase, diminish, weaken,reduce, inhibit, or mask the activity of an interferon, directly orindirectly. An example of directly affecting interferon involves, insome embodiments, an interferon-modulating polypeptide that specificallybinds to the interferon. Determination of which molecules possess thisactivity may be achieved using assays familiar to those of skill in theart. For example, transfer of genes encoding products that modulateinterferon, or variants thereof, into cells that are induced forinterferon activity compared to cells with such transfer of genes mayidentify, by virtue of different levels of an interferon response, thosemolecules having a interferon-modulating function.

It is specifically contemplated that a modulator may be a molecule thataffects the expression proteinaceous compositions involved in thetargeted molecule's pathway, such as by binding an interferon-encodingtranscript. Determination of which molecules are suitable modulators ofinterferon, IL-1β, TNF, or other molecules of therapeutic benefit may beachieved using assays familiar to those of skill in the art-some ofwhich are disclosed herein—and may include, for example, the use ofnative and/or recombinant viral proteins.

2. Variants of Viral Polypeptides

Amino acid sequence variants of the polypeptides of the presentinvention can be substitutional, insertional or deletion variants. Amutation in a gene encoding a viral polypeptide may affect 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200,210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475,500 or more non-contiguous or contiguous amino acids of the polypeptide,as compared to wild-type. Various polypeptides encoded by Vaccinia Virusmay be identified by reference to Rosel et al. (1986), Goebel et al.(1990) and GenBank Accession Number NC_001559, each of which isincorporated herein by reference.

Deletion variants lack one or more residues of the native or wild-typeprotein. Individual residues can be deleted or all or part of a domain(such as a catalytic or binding domain) can be deleted. A stop codon maybe introduced (by substitution or insertion) into an encoding nucleicacid sequence to generate a truncated protein. Insertional mutantstypically involve the addition of material at a non-terminal point inthe polypeptide. This may include the insertion of an immunoreactiveepitope or simply one or more residues. Terminal additions, calledfusion proteins, may also be generated.

Substitutional variants typically contain the exchange of one amino acidfor another at one or more sites within the protein, and may be designedto modulate one or more properties of the polypeptide, with or withoutthe loss of other functions or properties. Substitutions may beconservative, that is, one amino acid is replaced with one of similarshape and charge. Conservative substitutions are well known in the artand include, for example, the changes of: alanine to serine; arginine tolysine; asparagine to glutamine or histidine; aspartate to glutamate;cysteine to serine; glutamine to asparagine; glutamate to aspartate;glycine to proline; histidine to asparagine or glutamine; isoleucine toleucine or valine; leucine to valine or isoleucine; lysine to arginine;methionine to leucine or isoleucine; phenylalanine to tyrosine, leucineor methionine; serine to threonine; threonine to serine; tryptophan totyrosine; tyrosine to tryptophan or phenylalanine; and valine toisoleucine or leucine. Alternatively, substitutions may benon-conservative such that a function or activity of the polypeptide isaffected. Non-conservative changes typically involve substituting aresidue with one that is chemically dissimilar, such as a polar orcharged amino acid for a nonpolar or uncharged amino acid, and viceversa.

The term “functionally equivalent codon” is used herein to refer tocodons that encode the same amino acid, such as the six codons forarginine or serine, and also refers to codons that encode biologicallyequivalent amino acids (see Table 1, below).

It also will be understood that amino acid and nucleic acid sequencesmay include additional residues, such as additional N- or C-terminalamino acids or 5′ or 3′ sequences, and yet still be essentially as setforth in one of the sequences disclosed herein, so long as the sequencemeets the criteria set forth above, including the maintenance ofbiological protein activity where protein expression is concerned. Theaddition of terminal sequences particularly applies to nucleic acidsequences that may, for example, include various non-coding sequencesflanking either of the 5′ or 3′ portions of the coding region or mayinclude various internal sequences, i.e., introns, which are known tooccur within genes.

TABLE 1 Codon Table Amino Acids Codons Alanine Ala A GCA GCC GCG GCUCysteine Cys C UGC UGU  Aspartic acid Asp D GAC  GAU Glutamic acid Glu EGAA GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGUHistidine His H CAC CAU  Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine Leu L UUA UUG CUA CUC CUG CUU Methionine Met M AUGAsparagine Asn N AAC  AAU Proline Pro P CCA CCC CCG CCU Glutamine Gln QCAA  CAG Arginine Arg R AGA AGG CGA CGC CGG CGU Serine Ser S AGC AGU UCAUCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine Val V GUA GUC GUG GUUTryptophan Trp W UGG Tyrosine Tyr Y UAC UAU

The following is a discussion based upon changing of the amino acids ofa protein to create an equivalent, or even an improved,second-generation molecule. For example, certain amino acids may besubstituted for other amino acids in a protein structure withoutappreciable loss of interactive binding capacity with structures suchas, for example, antigen-binding regions of antibodies or binding siteson substrate molecules. Since it is the interactive capacity and natureof a protein that defines that protein's biological functional activity,certain amino acid substitutions can be made in a protein sequence, andin its underlying DNA coding sequence, and nevertheless produce aprotein with like properties. It is thus contemplated by the inventorsthat various changes may be made in the DNA sequences of genes withoutappreciable loss of their biological utility or activity, as discussedbelow. Table 1 shows the codons that encode particular amino acids.

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982). It is accepted thatthe relative hydropathic character of the amino acid contributes to thesecondary structure of the resultant protein, which in turn defines theinteraction of the protein with other molecules, for example, enzymes,substrates, receptors, DNA, antibodies, antigens, and the like.

It also is understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein. As detailed in U.S. Pat. No. 4,554,101, thefollowing hydrophilicity values have been assigned to amino acidresidues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate(+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine(0); threonine (−0.4); proline (−0.5±1); alanine (−0.5);histidine*−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5);leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine(−2.5); tryptophan (−3.4).

It is understood that an amino acid can be substituted for anotherhaving a similar hydrophilicity value and still produce a biologicallyequivalent and immunologically equivalent protein. In such changes, thesubstitution of amino acids whose hydrophilicity values are within ±2 ispreferred, those that are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

As outlined above, amino acid substitutions generally are based on therelative similarity of the amino acid side-chain substituents, forexample, their hydrophobicity, hydrophilicity, charge, size, and thelike. Exemplary substitutions that take into consideration the variousforegoing characteristics are well known to those of skill in the artand include: arginine and lysine; glutamate and aspartate; serine andthreonine; glutamine and asparagine; and valine, leucine and isoleucine.

IV. NUCLEIC ACID MOLECULES

A. Polynucleotides Encoding Native Proteins or Modified Proteins

The present invention concerns polynucleotides, isolatable from cells,that are capable of expressing all or part of a protein or polypeptide.In some embodiments of the invention, it concerns a viral genome thathas been specifically mutated to generate a virus that lacks certainfunctional viral polypeptides. The polynucleotides may encode a peptideor polypeptide containing all or part of a viral amino acid sequence orthey be engineered so they do not encode such a viral polypeptide orencode a viral polypeptide having at least one function or activityreduced, diminished, or absent. Recombinant proteins can be purifiedfrom expressing cells to yield active proteins. The genome, as well asthe definition of the coding regions of vaccinia virus may be found inRosel et al. (1986), Goebel et al. (1990) and/or GenBank AccessionNumber NC_00159, each of which is incorporated herein by reference.

As used herein, the term “DNA segment” refers to a DNA molecule that hasbeen isolated free of total genomic DNA of a particular species.Therefore, a DNA segment encoding a polypeptide refers to a DNA segmentthat contains wild-type, polymorphic, or mutant polypeptide-codingsequences yet is isolated away from, or purified free from, totalmammalian or human genomic DNA. Included within the term “DNA segment”are a polypeptide or polypeptides, DNA segments smaller than apolypeptide, and recombinant vectors, including, for example, plasmids,cosmids, phage, viruses, and the like.

As used in this application, the term “poxvirus polynucleotide” refersto a nucleic acid molecule encoding a poxvirus polypeptide that has beenisolated free of total genomic nucleic acid. Similarly, a “vacciniavirus polynucleotide” refers to a nucleic acid molecule encoding avaccinia virus polypeptide that has been isolated free of total genomicnucleic acid. A “poxvirus genome” or a “vaccinia virus genome” refers toa nucleic acid molecule that can be provided to a host cell to yield aviral particle, in the presence or absence of a helper virus. The genomemay or may have not been recombinantly mutated as compared to wild-typevirus.

The term “cDNA” is intended to refer to DNA prepared using messenger RNA(mRNA) as template. The advantage of using a cDNA, as opposed to genomicDNA or DNA polymerized from a genomic, non- or partially-processed RNAtemplate, is that the cDNA primarily contains coding sequences of thecorresponding protein. There may be times when the full or partialgenomic sequence is preferred, such as where the non-coding regions arerequired for optimal expression or where non-coding regions such asintrons are to be targeted in an antisense strategy.

It also is contemplated that a particular polypeptide from a givenspecies may be represented by natural variants that have slightlydifferent nucleic acid sequences but, nonetheless, encode the sameprotein (see Table 1 above).

Similarly, a polynucleotide comprising an isolated or purified wild-typeor mutant polypeptide gene refers to a DNA segment including wild-typeor mutant polypeptide coding sequences and, in certain aspects,regulatory sequences, isolated substantially away from other naturallyoccurring genes or protein encoding sequences. In this respect, the term“gene” is used for simplicity to refer to a functional protein,polypeptide, or peptide-encoding unit (including any sequences requiredfor proper transcription, post-translational modification, orlocalization). As will be understood by those in the art, thisfunctional term includes genomic sequences, cDNA sequences, and smallerengineered gene segments that express, or may be adapted to express,proteins, polypeptides, domains, peptides, fusion proteins, and mutants.A nucleic acid encoding all or part of a native or modified polypeptidemay contain a contiguous nucleic acid sequence encoding all or a portionof such a polypeptide of the following lengths: 10, 20, 30, 40, 50, 60,70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210,220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350,360, 370, 380, 390, 400, 410, 420, 430, 440, 441, 450, 460, 470, 480,490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620,630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760,770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900,910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030,1040, 1050, 1060, 1070, 1080, 1090, 1095, 1100, 1500, 2000, 2500, 3000,3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 9000, 10000,or more nucleotides, nucleosides, or base pairs.

In particular embodiments, the invention concerns isolated DNA segmentsand recombinant vectors incorporating DNA sequences that encode awild-type or mutant poxvirus polypeptide or peptide that includes withinits amino acid sequence a contiguous amino acid sequence in accordancewith, or essentially corresponding to a native polypeptide. Thus, anisolated DNA segment or vector containing a DNA segment may encode, forexample, a TNF modulator or TNF-modulating polypeptide that can inhibitor reduce TNF activity. The term “recombinant” may be used inconjunction with a polypeptide or the name of a specific polypeptide,and this generally refers to a polypeptide produced from a nucleic acidmolecule that has been manipulated in vitro or that is the replicatedproduct of such a molecule.

In other embodiments, the invention concerns isolated DNA segments andrecombinant vectors incorporating DNA sequences that encode apolypeptide or peptide that includes within its amino acid sequence acontiguous amino acid sequence in accordance with, or essentiallycorresponding to the polypeptide.

The nucleic acid segments used in the present invention, regardless ofthe length of the coding sequence itself, may be combined with othernucleic acid sequences, such as promoters, polyadenylation signals,additional restriction enzyme sites, multiple cloning sites, othercoding segments, and the like, such that their overall length may varyconsiderably. It is therefore contemplated that a nucleic acid fragmentof almost any length may be employed, with the total length preferablybeing limited by the ease of preparation and use in the intendedrecombinant DNA protocol.

It is contemplated that the nucleic acid constructs of the presentinvention may encode full-length polypeptide from any source or encode atruncated version of the polypeptide, for example a truncated vacciniavirus polypeptide, such that the transcript of the coding regionrepresents the truncated version. The truncated transcript may then betranslated into a truncated protein. Alternatively, a nucleic acidsequence may encode a full-length polypeptide sequence with additionalheterologous coding sequences, for example to allow for purification ofthe polypeptide, transport, secretion, post-translational modification,or for therapeutic benefits such as targeting or efficacy. As discussedabove, a tag or other heterologous polypeptide may be added to themodified polypeptide-encoding sequence, wherein “heterologous” refers toa polypeptide that is not the same as the modified polypeptide.

In a non-limiting example, one or more nucleic acid constructs may beprepared that include a contiguous stretch of nucleotides identical toor complementary to the a particular gene, such as the B18R gene. Anucleic acid construct may be at least 20, 30, 40, 50, 60, 70, 80, 90,100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 400,500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000,7,000, 8,000, 9,000, 10,000, 15,000, 20,000, 30,000, 50,000, 100,000,250,000, 500,000, 750,000, to at least 1,000,000 nucleotides in length,as well as constructs of greater size, up to and including chromosomalsizes (including all intermediate lengths and intermediate ranges),given the advent of nucleic acids constructs such as a yeast artificialchromosome are known to those of ordinary skill in the art. It will bereadily understood that “intermediate lengths” and “intermediateranges,” as used herein, means any length or range including or betweenthe quoted values (i.e., all integers including and between suchvalues).

The DNA segments used in the present invention encompass biologicallyfunctional equivalent modified polypeptides and peptides, for example, amodified gelonin toxin. Such sequences may arise as a consequence ofcodon redundancy and functional equivalency that are known to occurnaturally within nucleic acid sequences and the proteins thus encoded.Alternatively, functionally equivalent proteins or peptides may becreated via the application of recombinant DNA technology, in whichchanges in the protein structure may be engineered, based onconsiderations of the properties of the amino acids being exchanged.Changes designed by human may be introduced through the application ofsite-directed mutagenesis techniques, e.g., to introduce improvements tothe antigenicity of the protein, to reduce toxicity effects of theprotein in vivo to a subject given the protein, or to increase theefficacy of any treatment involving the protein.

In certain other embodiments, the invention concerns isolated DNAsegments and recombinant vectors that include within their sequence acontiguous nucleic acid sequence from that shown in sequences identifiedherein (and/or incorporated by reference). Such sequences, however, maybe mutated to yield a protein product whose activity is altered withrespect to wild-type.

It also will be understood that this invention is not limited to theparticular nucleic acid and amino acid sequences of these identifiedsequences. Recombinant vectors and isolated DNA segments may thereforevariously include the poxvirus-coding regions themselves, coding regionsbearing selected alterations or modifications in the basic codingregion, or they may encode larger polypeptides that nevertheless includepoxvirus-coding regions or may encode biologically functional equivalentproteins or peptides that have variant amino acids sequences.

The DNA segments of the present invention encompass biologicallyfunctional equivalent poxvirus proteins and peptides. Such sequences mayarise as a consequence of codon redundancy and functional equivalencythat are known to occur naturally within nucleic acid sequences and theproteins thus encoded. Alternatively, functionally equivalent proteinsor peptides may be created via the application of recombinant DNAtechnology, in which changes in the protein structure may be engineered,based on considerations of the properties of the amino acids beingexchanged. Changes designed by man may be introduced through theapplication of site-directed mutagenesis techniques, e.g., to introduceimprovements to the antigenicity of the protein.

B. Mutagenesis of Poxvirus Polynucleotides

In various embodiments, the poxvirus polynucleotide may be altered ormutagenized. Alterations or mutations may include insertions, deletions,point mutations, inversions, and the like and may result in themodulation, activation and/or inactivation of certain pathways ormolecular mechanisms, as well as altering the function, location, orexpression of a gene product, in particular rendering a gene productnon-functional. Where employed, mutagenesis of a polynucleotide encodingall or part of a Poxvirus may be accomplished by a variety of standard,mutagenic procedures (Sambrook et al., 1989). Mutation is the processwhereby changes occur in the quantity or structure of an organism.Mutation can involve modification of the nucleotide sequence of a singlegene, blocks of genes or whole chromosome. Changes in single genes maybe the consequence of point mutations which involve the removal,addition or substitution of a single nucleotide base within a DNAsequence, or they may be the consequence of changes involving theinsertion or deletion of large numbers of nucleotides.

Mutations may be induced following exposure to chemical or physicalmutagens. Such mutation-inducing agents include ionizing radiation,ultraviolet light and a diverse array of chemical such as alkylatingagents and polycyclic aromatic hydrocarbons all of which are capable ofinteracting either directly or indirectly (generally following somemetabolic biotransformations) with nucleic acids. The DNA damage inducedby such agents may lead to modifications of base sequence when theaffected DNA is replicated or repaired and thus to a mutation. Mutationalso can be site-directed through the use of particular targetingmethods.

1. Random Mutagenesis

a. Insertional Mutagenesis

Insertional mutagenesis is based on the inactivation of a gene viainsertion of a known DNA fragment. Because it involves the insertion ofsome type of DNA fragment, the mutations generated are generallyloss-of-function, rather than gain-of-function mutations. However, thereare several examples of insertions generating gain-of-functionmutations. Insertion mutagenesis has been very successful in bacteriaand Drosophila (Cooley et al., 1988) and recently has become a powerfultool in corn (Arabidopsis; (Marks et al., 1991; Koncz et al. 1990); andAntirrhinum (Sommer et al., 1990). Insertional mutagenesis may beaccomplished using standard molecular biology techniques.

b. Chemical Mutagenesis

Chemical mutagenesis offers certain advantages, such as the ability tofind a full range of mutations with degrees of phenotypic severity, andis facile and inexpensive to perform. The majority of chemicalcarcinogens produce mutations in DNA. Benzo[a]pyrene, N-acetoxy-2-acetylaminofluorene and aflotoxin B1 cause GC to TA transversions in bacteriaand mammalian cells. Benzo[a]pyrene also can produce base substitutionssuch as AT to TA. N-nitroso compounds produce GC to AT transitions.Alkylation of the O4 position of thymine induced by exposure ton-nitrosoureas results in TA to CG transitions.

c. Radiation Mutagenesis

Biological molecules are degraded by ionizing radiation. Adsorption ofthe incident energy leads to the formation of ions and free radicals,and breakage of some covalent bonds. Susceptibility to radiation damageappears quite variable between molecules, and between differentcrystalline forms of the same molecule. It depends on the totalaccumulated dose, and also on the dose rate (as once free radicals arepresent, the molecular damage they cause depends on their naturaldiffusion rate and thus upon real time). Damage is reduced andcontrolled by making the sample as cold as possible. Ionizing radiationcauses DNA damage, generally proportional to the dose rate.

In the present invention, the term “ionizing radiation” means radiationcomprising particles or photons that have sufficient energy or canproduce sufficient energy to produce ionization (gain or loss ofelectrons). An exemplary and preferred ionizing radiation is anx-radiation. The amount of ionizing radiation needed in a given cell orfor a particular molecule generally depends upon the nature of that cellor molecule and the nature of the mutation target. Means for determiningan effective amount of radiation are well known in the art.

d. In Vitro Scanning Mutagenesis

Random mutagenesis also may be introduced using error prone PCR. Therate of mutagenesis may be increased by performing PCR in multiple tubeswith dilutions of templates.

One particularly useful mutagenesis technique is alanine scanningmutagenesis in which a number of residues are substituted individuallywith the amino acid alanine so that the effects of losing side-chaininteractions can be determined, while minimizing the risk of large-scaleperturbations in protein conformation (Cunningham et al., 1989).

In vitro scanning saturation mutagenesis provides a rapid method forobtaining a large amount of structure-function information including (i)identification of residues that modulate ligand binding specificity,(ii) a better understanding of ligand binding based on theidentification of those amino acids that retain activity and those thatabolish activity at a given location, (iii) an evaluation of the overallplasticity of an active site or protein subdomain, (iv) identificationof amino acid substitutions that result in increased binding.

2. Site-Directed Mutagenesis

Structure-guided site-specific mutagenesis represents a powerful toolfor the dissection and engineering of protein-ligand interactions(Wells, 1996; Braisted et al., 1996). The technique provides for thepreparation and testing of sequence variants by introducing one or morenucleotide sequence changes into a selected DNA.

Site-specific mutagenesis uses specific oligonucleotide sequences whichencode the DNA sequence of the desired mutation, as well as a sufficientnumber of adjacent, unmodified nucleotides. In this way, a primersequence is provided with sufficient size and complexity to form astable duplex on both sides of the deletion junction being traversed. Aprimer of about 17 to 25 nucleotides in length is preferred, with about5 to 10 residues on both sides of the junction of the sequence beingaltered.

The technique typically employs a bacteriophage vector that exists inboth a single-stranded and double-stranded form. Vectors useful insite-directed mutagenesis include vectors such as the M13 phage. Thesephage vectors are commercially available and their use is generally wellknown to those skilled in the art. Double-stranded plasmids are alsoroutinely employed in site-directed mutagenesis, which eliminates thestep of transferring the gene of interest from a phage to a plasmid.

In general, one first obtains a single-stranded vector, or melts twostrands of a double-stranded vector, which includes within its sequencea DNA sequence encoding the desired protein or genetic element. Anoligonucleotide primer bearing the desired mutated sequence,synthetically prepared, is then annealed with the single-stranded DNApreparation, taking into account the degree of mismatch when selectinghybridization conditions. The hybridized product is subjected to DNApolymerizing enzymes such as E. coli polymerase I (Klenow fragment) inorder to complete the synthesis of the mutation-bearing strand. Thus, aheteroduplex is formed, wherein one strand encodes the originalnon-mutated sequence, and the second strand bears the desired mutation.This heteroduplex vector is then used to transform appropriate hostcells, such as E. coli cells, and clones are selected that includerecombinant vectors bearing the mutated sequence arrangement.

Comprehensive information on the functional significance and informationcontent of a given residue of protein can best be obtained by saturationmutagenesis in which all 19 amino acid substitutions are examined. Theshortcoming of this approach is that the logistics of multiresiduesaturation mutagenesis are daunting (Warren et al., 1996, Zeng et al.,1996; Burton and Barbas, 1994; Yelton et al., 1995; Hilton et al.,1996). Hundreds, and possibly even thousands, of site specific mutantsmust be studied. However, improved techniques make production and rapidscreening of mutants much more straightforward. See also, U.S. Pat. Nos.5,798,208 and 5,830,650, for a description of “walk-through”mutagenesis. Other methods of site-directed mutagenesis are disclosed inU.S. Pat. Nos. 5,220,007; 5,284,760; 5,354,670; 5,366,878; 5,389,514;5,635,377; and 5,789,166.

C. Vectors

The term “vector” is used to refer to a carrier nucleic acid moleculeinto which an exogenous nucleic acid sequence can be inserted forintroduction into a cell where it can be replicated. A nucleic acidsequence can be “exogenous,” which means that it is foreign to the cellinto which the vector is being introduced or that the sequence ishomologous to a sequence in the cell but in a position within the hostcell nucleic acid in which the sequence is ordinarily not found. Vectorsinclude plasmids, cosmids, viruses (bacteriophage, animal viruses, andplant viruses), and artificial chromosomes (e.g., YACs). One of skill inthe art would be well equipped to construct a vector through standardrecombinant techniques, which are described in Sambrook et al. (1989)and Ausubel et al. (1994), both incorporated herein by reference. Inaddition to encoding a modified polypeptide such as modified gelonin, avector may encode non-modified polypeptide sequences such as a tag ortargeting molecule. Useful vectors encoding such fusion proteins includepIN vectors (Inouye et al., 1985), vectors encoding a stretch ofhistidines, and pGEX vectors, for use in generating glutathioneS-transferase (GST) soluble fusion proteins for later purification andseparation or cleavage. A targeting molecule is one that directs themodified polypeptide to a particular organ, tissue, cell, or otherlocation in a subject's body.

The term “expression vector” refers to a vector containing a nucleicacid sequence coding for at least part of a gene product capable ofbeing transcribed. In some cases, RNA molecules are then translated intoa protein, polypeptide, or peptide. In other cases, these sequences arenot translated, for example, in the production of antisense molecules orribozymes. Expression vectors can contain a variety of “controlsequences,” which refer to nucleic acid sequences necessary for thetranscription and possibly translation of an operably linked codingsequence in a particular host organism. In addition to control sequencesthat govern transcription and translation, vectors and expressionvectors may contain nucleic acid sequences that serve other functions aswell and are described infra.

In accordance with the present invention, vaccinia virus is itself anexpression vector. There are other viral and non-viral vectors that mayalso be used to engineer the vaccinia viruses of the present invention.In one embodiment, such vectors may be engineered to express GM-CSF.

1. Promoters and Enhancers

A “promoter” is a control sequence that is a region of a nucleic acidsequence at which initiation and rate of transcription are controlled.It may contain genetic elements at which regulatory proteins andmolecules may bind such as RNA polymerase and other transcriptionfactors. The phrases “operatively positioned,” “operatively linked,”“under control,” and “under transcriptional control” mean that apromoter is in a correct functional location and/or orientation inrelation to a nucleic acid sequence to control transcriptionalinitiation and/or expression of that sequence. A promoter may or may notbe used in conjunction with an “enhancer,” which refers to a cis-actingregulatory sequence involved in the transcriptional activation of anucleic acid sequence.

A promoter may be one naturally associated with a gene or sequence, asmay be obtained by isolating the 5′ non-coding sequences locatedupstream of the coding segment and/or exon. Such a promoter can bereferred to as “endogenous.” Similarly, an enhancer may be one naturallyassociated with a nucleic acid sequence, located either downstream orupstream of that sequence. Alternatively, certain advantages will begained by positioning the coding nucleic acid segment under the controlof a recombinant or heterologous promoter, which refers to a promoterthat is not normally associated with a nucleic acid sequence in itsnatural environment. A recombinant or heterologous enhancer refers alsoto an enhancer not normally associated with a nucleic acid sequence inits natural environment. Such promoters or enhancers may includepromoters or enhancers of other genes, and promoters or enhancersisolated from any other prokaryotic, viral, or eukaryotic cell, andpromoters or enhancers not “naturally occurring,” i.e., containingdifferent elements of different transcriptional regulatory regions,and/or mutations that alter expression. In addition to producing nucleicacid sequences of promoters and enhancers synthetically, sequences maybe produced using recombinant cloning and/or nucleic acid amplificationtechnology, including PCR™, in connection with the compositionsdisclosed herein (see U.S. Pat. No. 4,683,202, U.S. Pat. No. 5,928,906,each incorporated herein by reference). Furthermore, it is contemplatedthe control sequences that direct transcription and/or expression ofsequences within non-nuclear organelles such as mitochondria,chloroplasts, and the like, can be employed as well.

Naturally, it may be important to employ a promoter and/or enhancer thateffectively directs the expression of the DNA segment in the cell type,organelle, and organism chosen for expression. In certain embodiments ofthe invention, the promoter is a vaccinia virus promoter that is activeduring the replication cycle of vaccinia virus. In particular, thepromoter may be the vaccinia virus late promoter—expression undercontrol of late promoter ties expression to later stage in replicationcycle, resulting in enhanced cancer-selectivity (because late geneexpression should be minimal or non-existent in normal tissues). Geneexpression could also be controlled under the synthetic early-latepromoter to maximize the duration and the level of gene expression.Mastrangelo et al., 1999, which is hereby incorporated by reference.

Those of skill in the art of molecular biology generally know the use ofpromoters, enhancers, and cell type combinations for protein expression,for example, see Sambrook et al. (1989), incorporated herein byreference. The promoters employed may be constitutive, tissue-specific,inducible, and/or useful under the appropriate conditions to direct highlevel expression of the introduced DNA segment, such as is advantageousin the large-scale production of recombinant proteins and/or peptides.The promoter may be heterologous or endogenous. Particular promoters arethose that can be active in the cytoplasm because the virus replicatesin the cytoplasm.

Table 2 lists several elements/promoters that may be employed, in thecontext of certain embodiments of the present invention, to regulate theexpression of a gene. This list is not intended to be exhaustive of allthe possible elements involved in the promotion of expression but,merely, to be exemplary thereof. Table 3 provides examples of inducibleelements, which are regions of a nucleic acid sequence that can beactivated in response to a specific stimulus.

TABLE 2 Promoter and/or Enhancer Promoter/Enhancer ReferencesImmunoglobulin Heavy Chain Banerji et al., 1983; Gilles et al., 1983;Grosschedl et al., 1985; Atchinson et al., 1986, 1987; Imler et al.,1987; Weinberger et al., 1984; Kiledjian et al., 1988; Porton et al.;1990 Immunoglobulin Light Chain Queen et al., 1983; Picard et al., 1984T-Cell Receptor Luria et al., 1987; Winoto et al., 1989; Redondo et al.;1990 HLA DQ a and/or DQ β Sullivan et al., 1987 β-Interferon Goodbournet al., 1986; Fujita et al., 1987; Goodbourn et al., 1988 Interleukin-2Greene et al., 1989 Interleukin-2 Receptor Greene et al., 1989; Lin etal., 1990 MHC Class II 5 Koch et al., 1989 MHC Class II HLA-DRa Shermanet al., 1989 β-Actin Kawamoto et al., 1988; Ng et al.; 1989 MuscleCreatine Kinase (MCK) Jaynes et al., 1988; Horlick et al., 1989; Johnsonet al., 1989 Prealbumin (Transthyretin) Costa et al., 1988 Elastase IOmitz et al., 1987 Metallothionein (MTII) Karin et al., 1987; Culotta etal., 1989 Collagenase Pinkert et al., 1987; Angel et al., 1987 AlbuminPinkert et al., 1987; Tronche et al., 1989, 1990 α-Fetoprotein Godboutet al., 1988; Campere et al., 1989 γ-Globin Bodine et al., 1987;Perez-Stable et al., 1990 β-Globin Trudel et al., 1987 c-fos Cohen etal., 1987 c-HA-ras Triesman, 1986; Deschamps et al., 1985 Insulin Edlundet al., 1985 Neural Cell Adhesion Molecule Hirsh et al., 1990 (NCAM)α₁-Antitrypain Latimer et al., 1990 H2B (TH2B) Histone Hwang et al.,1990 Mouse and/or Type I Collagen Ripe et al., 1989 Glucose-RegulatedProteins (GRP94 Chang et al., 1989 and GRP78) Rat Growth Hormone Larsenet al., 1986 Human Serum Amyloid A (SAA) Edbrooke et al., 1989 TroponinI (TN I) Yutzey et al., 1989 Platelet-Derived Growth Factor Pech et al.,1989 (PDGF) Duchenne Muscular Dystrophy Klamut et al., 1990 SV40 Banerjiet al., 1981; Moreau et al., 1981; Sleigh et al., 1985; Firak et al.,1986; Herr et al., 1986; Imbra et al., 1986; Kadesch et al., 1986; Wanget al., 1986; Ondek et al., 1987; Kuhl et al., 1987; Schaffner et al.,1988 Polyoma Swartzendruber et al., 1975; Vasseur et al., 1980; Katinkaet al., 1980, 1981; Tyndell et al., 1981; Dandolo et al., 1983; deVilliers et al., 1984; Hen et al., 1986; Satake et al., 1988; Campbellet al., 1988 Retroviruses Kriegler et al., 1982, 1983; Levinson et al.,1982; Kriegler et al., 1983, 1984a, b, 1988; Bosze et al., 1986;Miksicek et al., 1986; Celander et al., 1987; Thiesen et al., 1988;Celander et al., 1988; Chol et al., 1988; Reisman et al., 1989 PapillomaVirus Campo et al., 1983; Lusky et al., 1983; Spandidos and Wilkie,1983; Spalholz et al., 1985; Lusky et al., 1986; Cripe et al., 1987;Gloss et al., 1987; Hirochika et al., 1987; Stephens et al., 1987Hepatitis B Virus Bulla et al., 1986; Jameel et al., 1986; Shaul et al.,1987; Spandau et al., 1988; Vannice et al., 1988 Human ImmunodeficiencyVirus Muesing et al., 1987; Hauber et al., 1988; Jakobovits et al.,1988; Feng et al., 1988; Takebe et al., 1988; Rosen et al., 1988;Berkhout et al., 1989; Laspia et al., 1989; Sharp et al., 1989; Braddocket al., 1989 Cytomegalovirus (CMV) Weber et al., 1984; Boshart et al.,1985; Foecking et al., 1986 Gibbon Ape Leukemia Virus Holbrook et al.,1987; Quinn et al., 1989

TABLE 3 Inducible Elements Element Inducer References MT II PhorbolEster (TFA) Palmiter et al., 1982; Heavy metals Haslinger et al., 1985;Searle et al., 1985; Stuart et al., 1985; Imagawa et al., 1987, Karin etal., 1987; Angel et al., 1987b; McNeall et al., 1989 MMTV (mouse mammaryGlucocorticoids Huang et al., 1981; Lee et tumor virus) al., 1981;Majors et al., 1983; Chandler et al., 1983; Lee et al., 1984; Ponta etal., 1985; Sakai et al., 1988 β-Interferon poly(rI)x Tavernier et al.,1983 poly(rc) Adenovirus 5 E2 ElA Imperiale et al., 1984 CollagenasePhorbol Ester (TPA) Angel et al., 1987a Stromelysin Phorbol Ester (TPA)Angel et al., 1987b SV40 Phorbol Ester (TPA) Angel et al., 1987b MurineMX Gene Interferon, Newcastle Disease Hug et al., 1988 Virus GRP78 GeneA23187 Resendez et al., 1988 α-2-Macroglobulin IL-6 Kunz et al., 1989Vimentin Serum Rittling et al., 1989 MHC Class I Gene H-2κb InterferonBlanar et al., 1989 HSP70 ElA, SV40 Large T Antigen Taylor et al., 1989,1990a, 1990b Proliferin Phorbol Ester-TPA Mordacq et al., 1989 TumorNecrosis Factor PMA Hensel et al., 1989 Thyroid Stimulating HormoneThyroid Hormone Chatterjee et al., 1989 α Gene

The identity of tissue-specific promoters or elements, as well as assaysto characterize their activity, is well known to those of skill in theart. Examples of such regions include the human LIMK2 gene (Nomoto etal. 1999), the somatostatin receptor 2 gene (Kraus et al., 1998), murineepididymal retinoic acid-binding gene (Lareyre et al., 1999), human CD4(Zhao-Emonet et al., 1998), mouse alpha2 (XI) collagen (Tsumaki, et al.,1998), D1A dopamine receptor gene (Lee, et al., 1997), insulin-likegrowth factor II (Wu et al., 1997), human platelet endothelial celladhesion molecule-1 (Almendro et al., 1996), and the SM22α promoter.

Also contemplated as useful in the present invention are the dectin-1and dectin-2 promoters. Additional viral promoters, cellularpromoters/enhancers and inducible promoters/enhancers that could be usedin combination with the present invention are listed in Tables 2 and 3.Additionally any promoter/enhancer combination (as per the EukaryoticPromoter Data Base EPDB) could also be used to drive expression ofstructural genes encoding oligosaccharide processing enzymes, proteinfolding accessory proteins, selectable marker proteins or a heterologousprotein of interest. Alternatively, a tissue-specific promoter forcancer gene therapy (Table 4) or the targeting of tumors (Table 5) maybe employed with the nucleic acid molecules of the present invention.

TABLE 4 Candidate Tissue-Specific Promoters for Cancer Gene TherapyCancers in which promoter is Normal cells in which Tissue-specificpromoter active promoter is active Carcinoembryonic antigen Mostcolorectal carcinomas; 50% Colonic mucosa; gastric (CEA)* of lungcarcinomas; 40-50% of mucosa; lung epithelia; eccrine gastriccarcinomas; most sweat glands; cells in testes pancreatic carcinomas;many breast carcinomas Prostate-specific antigen Most prostatecarcinomas Prostate epithelium (PSA) Vasoactive intestinal peptideMajority of non-small cell lung Neurons; lymphocytes; mast (VIP) cancerscells; eosinophils Surfactant protein A (SP-A) Many lung adenocarcinomasType II pneumocytes; Clara cells Human achaete-scute Most small celllung cancers Neuroendocrine cells in lung homolog (hASH) Mucin-1(MUC1)** Most adenocarcinomas Glandular epithelial cells in (originatingfrom any tissue) breast and in respiratory, gastrointestinal, andgenitourinary tracts Alpha-fetoprotein Most hepatocellular carcinomas;Hepatocytes (under certain possibly many testicular cancers conditions);testis Albumin Most hepatocellular carcinomas Hepatocytes TyrosinaseMost melanomas Melanocytes; astrocytes; Schwann cells; some neuronsTyrosine-binding protein Most melanomas Melanocytes; astrocytes, (TRP)Schwann cells; some neurons Keratin 14 Presumably many squamous cellKeratinocytes carcinomas (e.g., Head and neck cancers) EBV LD-2 Manysquamous cell carcinomas Keratinocytes of upper of head and neckdigestive Keratinocytes of upper digestive tract Glial fibrillary acidicprotein Many astrocytomas Astrocytes (GFAP) Myelin basic protein (MBP)Many gliomas Oligodendrocytes Testis-specific angiotensin- Possibly manytesticular cancers Spermatazoa converting enzyme (Testis- specific ACE)Osteocalcin Possibly many osteosarcomas Osteoblasts

TABLE 5 Candidate Promoters for Use with a Tissue-Specific Targeting ofTumors Cancers in which Promoter is Normal cells in which Promoteractive Promoter is active E2F-regulated promoter Almost all cancersProliferating cells HLA-G Many colorectal carcinomas; Lymphocytes;monocytes; many melanomas; possibly many spermatocytes; trophoblastother cancers FasL Most melanomas; many Activated leukocytes: pancreaticcarcinomas; most neurons; endothelial cells; astrocytomas possibly manykeratinocytes; cells in other cancers immunoprivileged tissues; somecells in lungs, ovaries, liver, and prostate Myc-regulated promoter Mostlung carcinomas (both Proliferating cells (only some small cell andnon-small cell); cell-types): mammary epithelial most colorectalcarcinomas cells (including non- proliferating) MAGE-1 Many melanomas;some non- Testis small cell lung carcinomas; some breast carcinomas VEGF70% of all cancers (constitutive Cells at sites of overexpression inmany cancers) neovascularization (but unlike in tumors, expression istransient, less strong, and never constitutive) bFGF Presumably manydifferent Cells at sites of ischemia (but cancers, since bFGF expressionunlike tumors, expression is is induced by ischemic transient, lessstrong, and never conditions constitutive) COX-2 Most colorectalcarcinomas; Cells at sites of inflammation many lung carcinomas;possibly many other cancers IL-10 Most colorectal carcinomas; Leukocytesmany lung carcinomas; many squamous cell carcinomas of head and neck;possibly many other cancers GRP78/BiP Presumably many different Cells atsites of ishemia cancers, since GRP7S expression is induced bytumor-specific conditions CarG elements from Egr-1 Induced by ionizationradiation, Cells exposed to ionizing so conceivably most tumors uponradiation; leukocytes irradiation

2. Initiation Signals and Internal Ribosome Binding Sites

A specific initiation signal also may be required for efficienttranslation of coding sequences. These signals include the ATGinitiation codon or adjacent sequences. Exogenous translational controlsignals, including the ATG initiation codon, may need to be provided.One of ordinary skill in the art would readily be capable of determiningthis and providing the necessary signals. It is well known that theinitiation codon must be “in-frame” with the reading frame of thedesired coding sequence to ensure translation of the entire insert. Theexogenous translational control signals and initiation codons can beeither natural or synthetic. The efficiency of expression may beenhanced by the inclusion of appropriate transcription enhancerelements.

In certain embodiments of the invention, the use of internal ribosomeentry sites (IRES) elements are used to create multigene, orpolycistronic, messages. IRES elements are able to bypass the ribosomescanning model of 5′-methylated Cap dependent translation and begintranslation at internal sites (Pelletier and Sonenberg, 1988). IRESelements from two members of the picornavirus family (polio andencephalomyocarditis) have been described (Pelletier and Sonenberg,1988), as well an IRES from a mammalian message (Macejak and Sarnow,1991). IRES elements can be linked to heterologous open reading frames.Multiple open reading frames can be transcribed together, each separatedby an IRES, creating polycistronic messages. By virtue of the IRESelement, each open reading frame is accessible to ribosomes forefficient translation. Multiple genes can be efficiently expressed usinga single promoter/enhancer to transcribe a single message (see U.S. Pat.Nos. 5,925,565 and 5,935,819, herein incorporated by reference).

3. Multiple Cloning Sites

Vectors can include a multiple cloning site (MCS), which is a nucleicacid region that contains multiple restriction enzyme sites, any ofwhich can be used in conjunction with standard recombinant technology todigest the vector (see Carbonelli et al., 1999, Levenson et al., 1998,and Cocea, 1997, incorporated herein by reference). “Restriction enzymedigestion” refers to catalytic cleavage of a nucleic acid molecule withan enzyme that functions only at specific locations in a nucleic acidmolecule. Many of these restriction enzymes are commercially available.Use of such enzymes is widely understood by those of skill in the art.Frequently, a vector is linearized or fragmented using a restrictionenzyme that cuts within the MCS to enable exogenous sequences to beligated to the vector. “Ligation” refers to the process of formingphosphodiester bonds between two nucleic acid fragments, which may ormay not be contiguous with each other. Techniques involving restrictionenzymes and ligation reactions are well known to those of skill in theart of recombinant technology.

4. Splicing Sites

Most transcribed eukaryotic RNA molecules will undergo RNA splicing toremove introns from the primary transcripts. Vectors containing genomiceukaryotic sequences may require donor and/or acceptor splicing sites toensure proper processing of the transcript for protein expression. (SeeChandler et al., 1997, incorporated herein by reference.)

5. Termination Signals

The vectors or constructs of the present invention will generallycomprise at least one termination signal. A “termination signal” or“terminator” is comprised of the DNA sequences involved in specifictermination of an RNA transcript by an RNA polymerase. Thus, in certainembodiments a termination signal that ends the production of an RNAtranscript is contemplated. A terminator may be necessary in vivo toachieve desirable message levels.

In eukaryotic systems, the terminator region may also comprise specificDNA sequences that permit site-specific cleavage of the new transcriptso as to expose a polyadenylation site. This signals a specializedendogenous polymerase to add a stretch of about 200 A residues (polyA)to the 3′ end of the transcript. RNA molecules modified with this polyAtail appear to more stable and are translated more efficiently. Thus, inother embodiments involving eukaryotes, it is preferred that thatterminator comprises a signal for the cleavage of the RNA, and it ismore preferred that the terminator signal promotes polyadenylation ofthe message. The terminator and/or polyadenylation site elements canserve to enhance message levels and/or to minimize read through from thecassette into other sequences.

Terminators contemplated for use in the invention include any knownterminator of transcription described herein or known to one of ordinaryskill in the art, including but not limited to, for example, thetermination sequences of genes, such as for example the bovine growthhormone terminator or viral termination sequences, such as for examplethe SV40 terminator. In certain embodiments, the termination signal maybe a lack of transcribable or translatable sequence, such as due to asequence truncation.

6. Polyadenylation Signals

In expression, particularly eukaryotic expression, one will typicallyinclude a polyadenylation signal to effect proper polyadenylation of thetranscript. The nature of the polyadenylation signal is not believed tobe crucial to the successful practice of the invention, and/or any suchsequence may be employed. Preferred embodiments include the SV40polyadenylation signal and/or the bovine growth hormone polyadenylationsignal, convenient and/or known to function well in various targetcells. Polyadenylation may increase the stability of the transcript ormay facilitate cytoplasmic transport.

7. Origins of Replication

In order to propagate a vector in a host cell, it may contain one ormore origins of replication sites (often termed “ori”), which is aspecific nucleic acid sequence at which replication is initiated.Alternatively an autonomously replicating sequence (ARS) can be employedif the host cell is yeast.

8. Selectable and Screenable Markers

In certain embodiments of the invention, cells containing a nucleic acidconstruct of the present invention may be identified in vitro or in vivoby including a marker in the expression vector. Such markers wouldconfer an identifiable change to the cell permitting easy identificationof cells containing the expression vector. Generally, a selectablemarker is one that confers a property that allows for selection. Apositive selectable marker is one in which the presence of the markerallows for its selection, while a negative selectable marker is one inwhich its presence prevents its selection. An example of a positiveselectable marker is a drug resistance marker.

Usually the inclusion of a drug selection marker aids in the cloning andidentification of transformants, for example, genes that conferresistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin andhistidinol are useful selectable markers. In addition to markersconferring a phenotype that allows for the discrimination oftransformants based on the implementation of conditions, other types ofmarkers including screenable markers such as GFP, whose basis iscolorimetric analysis, are also contemplated. Alternatively, screenableenzymes such as herpes simplex virus thymidine kinase (tk) orchloramphenicol acetyltransferase (CAT) may be utilized. One of skill inthe art would also know how to employ immunologic markers, possibly inconjunction with FACS analysis. The marker used is not believed to beimportant, so long as it is capable of being expressed simultaneouslywith the nucleic acid encoding a gene product. Further examples ofselectable and screenable markers are well known to one of skill in theart.

D. Nucleic Acid Detection

In addition to their use in directing the expression of poxvirusproteins, polypeptides and/or peptides, the nucleic acid sequencesdisclosed herein have a variety of other uses. For example, they haveutility as probes or primers for embodiments involving nucleic acidhybridization. They may be used in diagnostic or screening methods ofthe present invention. Detection of nucleic acids encoding poxvirus orpoxvirus polypeptide modulators are encompassed by the invention.

1. Hybridization

The use of a probe or primer of between 13 and 100 nucleotides,preferably between 17 and 100 nucleotides in length, or in some aspectsof the invention up to 1-2 kilobases or more in length, allows theformation of a duplex molecule that is both stable and selective.Molecules having complementary sequences over contiguous stretchesgreater than 20 bases in length are generally preferred, to increasestability and/or selectivity of the hybrid molecules obtained. One willgenerally prefer to design nucleic acid molecules for hybridizationhaving one or more complementary sequences of 20 to 30 nucleotides, oreven longer where desired. Such fragments may be readily prepared, forexample, by directly synthesizing the fragment by chemical means or byintroducing selected sequences into recombinant vectors for recombinantproduction.

Accordingly, the nucleotide sequences of the invention may be used fortheir ability to selectively form duplex molecules with complementarystretches of DNAs and/or RNAs or to provide primers for amplification ofDNA or RNA from samples. Depending on the application envisioned, onewould desire to employ varying conditions of hybridization to achievevarying degrees of selectivity of the probe or primers for the targetsequence.

For applications requiring high selectivity, one will typically desireto employ relatively high stringency conditions to form the hybrids. Forexample, relatively low salt and/or high temperature conditions, such asprovided by about 0.02 M to about 0.10 M NaCl at temperatures of about50° C. to about 70° C. Such high stringency conditions tolerate little,if any, mismatch between the probe or primers and the template or targetstrand and would be particularly suitable for isolating specific genesor for detecting specific mRNA transcripts. It is generally appreciatedthat conditions can be rendered more stringent by the addition ofincreasing amounts of formamide.

For certain applications, for example, site-directed mutagenesis, it isappreciated that lower stringency conditions are preferred. Under theseconditions, hybridization may occur even though the sequences of thehybridizing strands are not perfectly complementary, but are mismatchedat one or more positions. Conditions may be rendered less stringent byincreasing salt concentration and/or decreasing temperature. Forexample, a medium stringency condition could be provided by about 0.1 to0.25 M NaCl at temperatures of about 37° C. to about 55° C., while a lowstringency condition could be provided by about 0.15 M to about 0.9 Msalt, at temperatures ranging from about 20° C. to about 55° C.Hybridization conditions can be readily manipulated depending on thedesired results.

In other embodiments, hybridization may be achieved under conditions of,for example, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl₂, 1.0 mMdithiothreitol, at temperatures between approximately 20° C. to about37° C. Other hybridization conditions utilized could includeapproximately 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, attemperatures ranging from approximately 40° C. to about 72° C.

In certain embodiments, it will be advantageous to employ nucleic acidsof defined sequences of the present invention in combination with anappropriate means, such as a label, for determining hybridization. Awide variety of appropriate indicator means are known in the art,including fluorescent, radioactive, enzymatic or other ligands, such asavidin/biotin, which are capable of being detected. In preferredembodiments, one may desire to employ a fluorescent label or an enzymetag such as urease, alkaline phosphatase or peroxidase, instead ofradioactive or other environmentally undesirable reagents. In the caseof enzyme tags, colorimetric indicator substrates are known that can beemployed to provide a detection means that is visibly orspectrophotometrically detectable, to identify specific hybridizationwith complementary nucleic acid containing samples.

In general, it is envisioned that the probes or primers described hereinwill be useful as reagents in solution hybridization, as in PCR™, fordetection of expression of corresponding genes, as well as inembodiments employing a solid phase. In embodiments involving a solidphase, the test DNA (or RNA) is adsorbed or otherwise affixed to aselected matrix or surface. This fixed, single-stranded nucleic acid isthen subjected to hybridization with selected probes under desiredconditions. The conditions selected will depend on the particularcircumstances (depending, for example, on the G+C content, type oftarget nucleic acid, source of nucleic acid, size of hybridizationprobe, etc.). Optimization of hybridization conditions for theparticular application of interest is well known to those of skill inthe art. After washing of the hybridized molecules to removenon-specifically bound probe molecules, hybridization is detected,and/or quantified, by determining the amount of bound label.Representative solid phase hybridization methods are disclosed in U.S.Pat. Nos. 5,843,663, 5,900,481 and 5,919,626. Other methods ofhybridization that may be used in the practice of the present inventionare disclosed in U.S. Pat. Nos. 5,849,481, 5,849,486 and 5,851,772. Therelevant portions of these and other references identified in thissection of the Specification are incorporated herein by reference.

2. Amplification of Nucleic Acids

Nucleic acids used as a template for amplification may be isolated fromcells, tissues or other samples according to standard methodologies(Sambrook et al., 1989). In certain embodiments, analysis is performedon whole cell or tissue homogenates or biological fluid samples withoutsubstantial purification of the template nucleic acid. The nucleic acidmay be genomic DNA or fractionated or whole cell RNA. Where RNA is used,it may be desired to first convert the RNA to a complementary DNA.

The term “primer,” as used herein, is meant to encompass any nucleicacid that is capable of priming the synthesis of a nascent nucleic acidin a template-dependent process. Typically, primers are oligonucleotidesfrom ten to twenty and/or thirty base pairs in length, but longersequences can be employed. Primers may be provided in double-strandedand/or single-stranded form, although the single-stranded form ispreferred.

Pairs of primers designed to selectively hybridize to nucleic acidscorresponding to sequences of genes identified herein are contacted withthe template nucleic acid under conditions that permit selectivehybridization. Depending upon the desired application, high stringencyhybridization conditions may be selected that will only allowhybridization to sequences that are completely complementary to theprimers. In other embodiments, hybridization may occur under reducedstringency to allow for amplification of nucleic acids contain one ormore mismatches with the primer sequences. Once hybridized, thetemplate-primer complex is contacted with one or more enzymes thatfacilitate template-dependent nucleic acid synthesis. Multiple rounds ofamplification, also referred to as “cycles,” are conducted until asufficient amount of amplification product is produced.

The amplification product may be detected or quantified. In certainapplications, the detection may be performed by visual means.Alternatively, the detection may involve indirect identification of theproduct via chemiluminescence, radioactive scintigraphy of incorporatedradiolabel or fluorescent label or even via a system using electricaland/or thermal impulse signals (Bellus, 1994).

A number of template dependent processes are available to amplify theoligonucleotide sequences present in a given template sample. One of thebest known amplification methods is the polymerase chain reaction(referred to as PCR™) which is described in detail in U.S. Pat. Nos.4,683,195, 4,683,202 and 4,800,159, and in Innis et al., 1988, each ofwhich is incorporated herein by reference in their entirety.

A reverse transcriptase PCR™ amplification procedure may be performed toquantify the amount of mRNA amplified. Methods of reverse transcribingRNA into cDNA are well known (see Sambrook et al., 1989). Alternativemethods for reverse transcription utilize thermostable DNA polymerases.These methods are described in WO 90/07641. Polymerase chain reactionmethodologies are well known in the art. Representative methods ofRT-PCR are described in U.S. Pat. No. 5,882,864.

Another method for amplification is ligase chain reaction (“LCR”),disclosed in European Application No. 320 308, incorporated herein byreference in its entirety. U.S. Pat. No. 4,883,750 describes a methodsimilar to LCR for binding probe pairs to a target sequence. A methodbased on PCR™ and oligonucleotide ligase assay (OLA), disclosed in U.S.Pat. No. 5,912,148, may also be used.

Alternative methods for amplification of target nucleic acid sequencesthat may be used in the practice of the present invention are disclosedin U.S. Pat. Nos. 5,843,650, 5,846,709, 5,846,783, 5,849,546, 5,849,497,5,849,547, 5,858,652, 5,866,366, 5,916,776, 5,922,574, 5,928,905,5,928,906, 5,932,451, 5,935,825, 5,939,291 and 5,942,391, GB ApplicationNo. 2 202 328, and in PCT Application No. PCT/US89/01025, each of whichis incorporated herein by reference in its entirety.

Qbeta Replicase, described in PCT Application No. PCT/US87/00880, mayalso be used as an amplification method in the present invention. Inthis method, a replicative sequence of RNA that has a regioncomplementary to that of a target is added to a sample in the presenceof an RNA polymerase. The polymerase will copy the replicative sequencewhich may then be detected.

An isothermal amplification method, in which restriction endonucleasesand ligases are used to achieve the amplification of target moleculesthat contain nucleotide 5′-[alpha-thio]-triphosphates in one strand of arestriction site may also be useful in the amplification of nucleicacids in the present invention (Walker et al., 1992). StrandDisplacement Amplification (SDA), disclosed in U.S. Pat. No. 5,916,779,is another method of carrying out isothermal amplification of nucleicacids which involves multiple rounds of strand displacement andsynthesis, i.e., nick translation.

Other nucleic acid amplification procedures include transcription-basedamplification systems (TAS), including nucleic acid sequence basedamplification (NASBA) and 3SR (Kwoh et al., 1989; PCT Application WO88/10315, incorporated herein by reference in their entirety). EuropeanApplication No. 329 822 disclose a nucleic acid amplification processinvolving cyclically synthesizing single-stranded RNA (“ssRNA”), ssDNA,and double-stranded DNA (dsDNA), which may be used in accordance withthe present invention.

PCT Application WO 89/06700 (incorporated herein by reference in itsentirety) disclose a nucleic acid sequence amplification scheme based onthe hybridization of a promoter region/primer sequence to a targetsingle-stranded DNA (“ssDNA”) followed by transcription of many RNAcopies of the sequence. This scheme is not cyclic, i.e., new templatesare not produced from the resultant RNA transcripts. Other amplificationmethods include “RACE” and “one-sided PCR” (Frohman, 1990; Ohara et al.,1989).

3. Detection of Nucleic Acids

Following any amplification, it may be desirable to separate theamplification product from the template and/or the excess primer. In oneembodiment, amplification products are separated by agarose,agarose-acrylamide or polyacrylamide gel electrophoresis using standardmethods (Sambrook et al., 1989). Separated amplification products may becut out and eluted from the gel for further manipulation. Using lowmelting point agarose gels, the separated band may be removed by heatingthe gel, followed by extraction of the nucleic acid.

Separation of nucleic acids may also be effected by chromatographictechniques known in art. There are many kinds of chromatography whichmay be used in the practice of the present invention, includingadsorption, partition, ion-exchange, hydroxylapatite, molecular sieve,reverse-phase, column, paper, thin-layer, and gas chromatography as wellas HPLC.

In certain embodiments, the amplification products are visualized. Atypical visualization method involves staining of a gel with ethidiumbromide and visualization of bands under UV light. Alternatively, if theamplification products are integrally labeled with radio- orfluorometrically-labeled nucleotides, the separated amplificationproducts can be exposed to x-ray film or visualized under theappropriate excitatory spectra.

In one embodiment, following separation of amplification products, alabeled nucleic acid probe is brought into contact with the amplifiedmarker sequence. The probe preferably is conjugated to a chromophore butmay be radiolabeled. In another embodiment, the probe is conjugated to abinding partner, such as an antibody or biotin, or another bindingpartner carrying a detectable moiety.

In particular embodiments, detection is by Southern blotting andhybridization with a labeled probe. The techniques involved in Southernblotting are well known to those of skill in the art (see Sambrook etal., 1989). One example of the foregoing is described in U.S. Pat. No.5,279,721, incorporated by reference herein, which discloses anapparatus and method for the automated electrophoresis and transfer ofnucleic acids. The apparatus permits electrophoresis and blottingwithout external manipulation of the gel and is ideally suited tocarrying out methods according to the present invention.

Other methods of nucleic acid detection that may be used in the practiceof the instant invention are disclosed in U.S. Pat. Nos. 5,840,873,5,843,640, 5,843,651, 5,846,708, 5,846,717, 5,846,726, 5,846,729,5,849,487, 5,853,990, 5,853,992, 5,853,993, 5,856,092, 5,861,244,5,863,732, 5,863,753, 5,866,331, 5,905,024, 5,910,407, 5,912,124,5,912,145, 5,919,630, 5,925,517, 5,928,862, 5,928,869, 5,929,227,5,932,413 and 5,935,791, each of which is incorporated herein byreference.

4. Other Assays

Other methods for genetic screening may be used within the scope of thepresent invention, for example, to detect mutations in genomic DNA, cDNAand/or RNA samples. Methods used to detect point mutations includedenaturing gradient gel electrophoresis (“DGGE”), restriction fragmentlength polymorphism analysis (“RFLP”), chemical or enzymatic cleavagemethods, direct sequencing of target regions amplified by PCR™ (seeabove), single-strand conformation polymorphism analysis (“SSCP”) andother methods well known in the art.

One method of screening for point mutations is based on RNase cleavageof base pair mismatches in RNA/DNA or RNA/RNA heteroduplexes. As usedherein, the term “mismatch” is defined as a region of one or moreunpaired or mispaired nucleotides in a double-stranded RNA/RNA, RNA/DNAor DNA/DNA molecule. This definition thus includes mismatches due toinsertion/deletion mutations, as well as single or multiple base pointmutations.

U.S. Pat. No. 4,946,773 describes an RNase A mismatch cleavage assaythat involves annealing single-stranded DNA or RNA test samples to anRNA probe, and subsequent treatment of the nucleic acid duplexes withRNase A. For the detection of mismatches, the single-stranded productsof the RNase A treatment, electrophoretically separated according tosize, are compared to similarly treated control duplexes. Samplescontaining smaller fragments (cleavage products) not seen in the controlduplex are scored as positive.

Other investigators have described the use of RNase I in mismatchassays. The use of RNase I for mismatch detection is described inliterature from Promega Biotech. Promega markets a kit containing RNaseI that is reported to cleave three out of four known mismatches. Othershave described using the MutS protein or other DNA-repair enzymes fordetection of single-base mismatches.

Alternative methods for detection of deletion, insertion or substitutionmutations that may be used in the practice of the present invention aredisclosed in U.S. Pat. Nos. 5,849,483, 5,851,770, 5,866,337, 5,925,525and 5,928,870, each of which is incorporated herein by reference in itsentirety.

E. Methods of Gene Transfer

Suitable methods for nucleic acid delivery to effect expression ofcompositions of the present invention are believed to include virtuallyany method by which a nucleic acid (e.g., DNA, including viral andnonviral vectors) can be introduced into an organelle, a cell, a tissueor an organism, as described herein or as would be known to one ofordinary skill in the art. Such methods include, but are not limited to,direct delivery of DNA such as by injection (U.S. Pat. Nos. 5,994,624,5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610,5,589,466 and 5,580,859, each incorporated herein by reference),including microinjection (Harlan and Weintraub, 1985; U.S. Pat. No.5,789,215, incorporated herein by reference); by electroporation (U.S.Pat. No. 5,384,253, incorporated herein by reference); by calciumphosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama,1987; Rippe et al., 1990); by using DEAE-dextran followed bypolyethylene glycol (Gopal, 1985); by direct sonic loading (Fechheimeret al., 1987); by liposome mediated transfection (Nicolau and Sene,1982; Fraley et al., 1979; Nicolau et al., 1987; Wong et al., 1980;Kaneda et al., 1989; Kato et al., 1991); by microprojectile bombardment(PCT Application Nos. WO 94/09699 and 95/06128; U.S. Pat. Nos.5,610,042; 5,322,783 5,563,055, 5,550,318, 5,538,877 and 5,538,880, andeach incorporated herein by reference); by agitation with siliconcarbide fibers (Kaeppler et al., 1990; U.S. Pat. Nos. 5,302,523 and5,464,765, each incorporated herein by reference); byAgrobacterium-mediated transformation (U.S. Pat. Nos. 5,591,616 and5,563,055, each incorporated herein by reference); or by PEG-mediatedtransformation of protoplasts (Omirulleh et al., 1993; U.S. Pat. Nos.4,684,611 and 4,952,500, each incorporated herein by reference); bydesiccation/inhibition-mediated DNA uptake (Potrykus et al., 1985).Through the application of techniques such as these, organelle(s),cell(s), tissue(s) or organism(s) may be stably or transientlytransformed.

5. Lipid Components and Moieties

In certain embodiments, the present invention concerns compositionscomprising one or more lipids associated with a nucleic acid, an aminoacid molecule, such as a peptide, or another small molecule compound. Inany of the embodiments discussed herein, the molecule may be either apoxvirus polypeptide or a poxvirus polypeptide modulator, for example anucleic acid encoding all or part of either a poxvirus polypeptide, oralternatively, an amino acid molecule encoding all or part of poxviruspolypeptide modulator. A lipid is a substance that is characteristicallyinsoluble in water and extractable with an organic solvent. Compoundsthan those specifically described herein are understood by one of skillin the art as lipids, and are encompassed by the compositions andmethods of the present invention. A lipid component and a non-lipid maybe attached to one another, either covalently or non-covalently.

A lipid may be naturally occurring or synthetic (i.e., designed orproduced by man). However, a lipid is usually a biological substance.Biological lipids are well known in the art, and include for example,neutral fats, phospholipids, phosphoglycerides, steroids, terpenes,lysolipids, glycosphingolipids, glucolipids, sulphatides, lipids withether and ester-linked fatty acids and polymerizable lipids, andcombinations thereof.

A nucleic acid molecule or amino acid molecule, such as a peptide,associated with a lipid may be dispersed in a solution containing alipid, dissolved with a lipid, emulsified with a lipid, mixed with alipid, combined with a lipid, covalently bonded to a lipid, contained asa suspension in a lipid or otherwise associated with a lipid. A lipid orlipid/poxvirus-associated composition of the present invention is notlimited to any particular structure. For example, they may also simplybe interspersed in a solution, possibly forming aggregates which are notuniform in either size or shape. In another example, they may be presentin a bilayer structure, as micelles, or with a “collapsed” structure. Inanother non-limiting example, a lipofectamine (Gibco BRL)-poxvirus orSuperfect (Qiagen)-poxvirus complex is also contemplated.

In certain embodiments, a lipid composition may comprise about 1%, about2%, about 3%, about 4% about 5%, about 6%, about 7%, about 8%, about 9%,about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%,about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%,about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%,about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%,about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%,about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%,about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100%,or any range derivable therein, of a particular lipid, lipid type ornon-lipid component such as a drug, protein, sugar, nucleic acids orother material disclosed herein or as would be known to one of skill inthe art. In a non-limiting example, a lipid composition may compriseabout 10% to about 20% neutral lipids, and about 33% to about 34% of acerebroside, and about 1% cholesterol. In another non-limiting example,a liposome may comprise about 4% to about 12% terpenes, wherein about 1%of the micelle is specifically lycopene, leaving about 3% to about 11%of the liposome as comprising other terpenes; and about 10% to about 35%phosphatidyl choline, and about 1% of a drug. Thus, it is contemplatedthat lipid compositions of the present invention may comprise any of thelipids, lipid types or other components in any combination or percentagerange.

V. GM-CSF

In a particular aspect of the invention, the vaccinia viruses will carrya gene encoding for GM-CSF. GM-CSF is granulocyte-macrophagecolony-stimulating factor, a substance that helps make more white bloodcells, especially granulocytes, macrophages, and cells that becomeplatelets. It is a cytokine that belongs to the family of drugs calledhematopoietic (blood-forming) agents, and is also known as sargramostim.GM-CSF was first cloned and sequence in 1985 by Cantrell et al. (1985).Human GM-CSF is a 144-amino acid glycoprotein encoded by a singleopen-reading frame with a predicted molecular mass of 16,293 daltons. Itexhibits a 69% nucleotide homology and 54% amino acid homology to mouseGM-CSF and exists as a single-copy gene.

GM-CSF is produced by a number of different cell types (includingactivated T cells, B cells, macrophages, mast cells, endothelial cellsand fibroblasts) in response to cytokine or immune and inflammatorystimuli. Besides granulocyte-macrophage progenitors, GM-CSF is also agrowth factor for erythroid, megakaryocyte and eosinophil progenitors.On mature hematopoietic cells, GM-CSF is a survival factor for andactivates the effector functions of granulocytes, monocytes/macrophagesand eosinophils. GM-CSF has also been reported to have a functional roleon non-hematopoietic cells. It can induce human endothelial cells tomigrate and proliferate.

GM-CSF is species specific and human GM-CSF has no biological effects onmouse cells. GM-CSF exerts its biological effects through binding tospecific cell surface receptors. The high affinity receptors requiredfor human GM-CSF signal transduction have been shown to be heterodimersconsisting of a GM-CSF-specific a chain and a common β chain that isshared by the high-affinity receptors for IL-3 and IL-5.

Although GM-CSF can stimulate the proliferation of a number of tumorcell lines, including osteogenic sarcoma, carcinoma and adenocarcinomacell lines, clinical trials of GM-CSF (alone or with otherimmunotherapies) are in progress for people with melanoma, leukemia,lymphoma, neuroblastoma, Kaposi sarcoma, mesothelioma, lung cancer,breast cancer, prostate cancer, colorectal cancer, brain tumors, kidneycancer and cervical cancer. Common side effects of GM-CSF includeflu-like symptoms (fever, headaches, muscle aches), rashes, facialflushing, and bone pain.

VI. OTHER HETEROLOGOUS GENES

In some embodiments, the vaccinia virus used in methods of the inventioncontains a nucleic acid sequence that expresses a heterologous sequencethat does not encode GM-CSF but encodes another heterologous sequence.In certain embodiments, the heterologous sequence encodes anothercytokine. Alternatively or additionally, the vaccinia virus may containa nucleic acid that encodes for IL-12, thymidine deaminase, TNF, and thelike. In addition, any gene product discussed herein may be encoded by anucleic acid contained within a vaccinia virus and used in methods ofthe invention.

VII. PHARMACEUTICAL FORMULATIONS, DELIVERY, AND TREATMENT REGIMENS

In an embodiment of the present invention, a method of treatment for ahyperproliferative disease, such as cancer, by the delivery of avaccinia virus, is contemplated. Examples of cancer contemplated fortreatment include lung cancer, head and neck cancer, breast cancer,pancreatic cancer, prostate cancer, renal cancer, uterine cancer, bonecancer, testicular cancer, cervical cancer, gastrointestinal cancer,lymphomas, pre-neoplastic lesions in the lung, colon cancer, melanoma,bladder cancer and any other cancers or tumors that may be treated.

An effective amount of the pharmaceutical composition, generally, isdefined as that amount sufficient to detectably and repeatedly toameliorate, reduce, minimize or limit the extent of the disease or itssymptoms. More rigorous definitions may apply, including elimination,eradication or cure of disease.

Preferably, patients will have adequate bone marrow function (defined asa peripheral absolute granulocyte count of >2,000/mm³ and a plateletcount of 100,000/mm³), adequate liver function (bilirubin <1.5 mg/dl)and adequate renal function (creatinine <1.5 mg/dl).

Cancer cells that may be treated by methods and compositions of theinvention include cells from the bladder, blood, bone, bone marrow,brain, breast, colon, esophagus, gastrointestine, gum, head, kidney,liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis,tongue, or uterus. In addition, the cancer may specifically be of thefollowing histological type, though it is not limited to these:neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant andspindle cell carcinoma; small cell carcinoma; papillary carcinoma;squamous cell carcinoma; lymphoepithelial carcinoma; basal cellcarcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillarytransitional cell carcinoma; adenocarcinoma; gastrinoma, malignant;cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellularcarcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoidcystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma,familial polyposis coli; solid carcinoma; carcinoid tumor, malignant;branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma;chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma;basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma;follicular adenocarcinoma; papillary and follicular adenocarcinoma;nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma;endometroid carcinoma; skin appendage carcinoma; apocrineadenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma;mucoepidermoid carcinoma; cystadenocarcinoma; papillarycystadenocarcinoma; papillary serous cystadenocarcinoma; mucinouscystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma;infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma;inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma;adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma,malignant; ovarian stromal tumor, malignant; thecoma, malignant;granulosa cell tumor, malignant; androblastoma, malignant; sertoli cellcarcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant;paraganglioma, malignant; extra-mammary paraganglioma, malignant;pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanoticmelanoma; superficial spreading melanoma; malig melanoma in giantpigmented nevus; epithelioid cell melanoma; blue nevus, malignant;sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma;liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonalrhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixedtumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma;carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant;phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant;dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii,malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma;hemangioendothelioma, malignant; Kaposi's sarcoma; hemangiopericytoma,malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma;chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma;giant cell tumor of bone; Ewing's sarcoma; odontogenic tumor, malignant;ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblasticfibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant;ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillaryastrocytoma; astroblastoma; glioblastoma; oligodendroglioma;oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma;ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactoryneurogenic tumor; meningioma, malignant; neurofibrosarcoma;neurilemmoma, malignant; granular cell tumor, malignant; malignantlymphoma; hodgkin's disease; hodgkin's; paragranuloma; malignantlymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse;malignant lymphoma, follicular; mycosis fungoides; other specifiednon-hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mastcell sarcoma; immunoproliferative small intestinal disease; leukemia;lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcomacell leukemia; myeloid leukemia; basophilic leukemia; eosinophilicleukemia; monocytic leukemia; mast cell leukemia; megakaryoblasticleukemia; myeloid sarcoma; and hairy cell leukemia.

The present invention contemplates methods for inhibiting or preventinglocal invasiveness and/or metastasis of any type of primary cancer. Forexample, the primary cancer may be melanoma, non-small cell lung,small-cell lung, lung, hepatocarcinoma, retinoblastoma, astrocytoma,glioblastoma, gum, tongue, leukemia, neuroblastoma, head, neck, breast,pancreatic, prostate, renal, bone, testicular, ovarian, mesothelioma,cervical, gastrointestinal, lymphoma, brain, colon, or bladder. Incertain embodiments of the present invention, the primary cancer is lungcancer. For example, the lung cancer may be non-small cell lungcarcinoma.

Moreover, the present invention can be used to prevent cancer or totreat pre-cancers or premalignant cells, including metaplasias,dysplasias, and hyperplasias. It may also be used to inhibit undesirablebut benign cells, such as squamous metaplasia, dysplasia, benignprostate hyperplasia cells, hyperplastic lesions, and the like. Theprogression to cancer or to a more severe form of cancer may be halted,disrupted, or delayed by methods of the invention involving GM-CSFpolypeptides or other polypeptide(s) encoded by a vaccinia virus, asdiscussed herein.

A. Administration

To kill cells, inhibit cell growth, inhibit metastasis, decrease tumoror tissue size and otherwise reverse or reduce the malignant phenotypeof tumor cells, using the methods and compositions of the presentinvention, one would generally contact a hyperproliferative cell withthe therapeutic compound such as a polypeptide or an expressionconstruct encoding a polypeptide. The routes of administration willvary, naturally, with the location and nature of the lesion, andinclude, e.g., intradermal, transdermal, parenteral, intravenous,intramuscular, intranasal, subcutaneous, regional, percutaneous,intratracheal, intraperitoneal, intraarterial, intravesical,intratumoral, inhalation, perfusion, lavage, direct injection, and oraladministration and formulation.

The present invention specifically concerns intravascular administrationof a vaccinia virus of the invention. The term “intravascular” isunderstood to refer to delivery into the vasculature of a patient,meaning into, within, or in a vessel or vessels of the patient. Incertain embodiments, the administration is into a vessel considered tobe a vein (intravenous), while in others administration is into a vesselconsidered to be an artery. Veins include, but are not limited to, theinternal jugular vein, a peripheral vein, a coronary vein, a hepaticvein, the portal vein, great saphenous vein, the pulmonary vein,superior vena cava, inferior vena cava, a gastric vein, a splenic vein,inferior mesenteric vein, superior mesenteric vein, cephalic vein,and/or femoral vein. Arteries include, but are not limited to, coronaryartery, pulmonary artery, brachial artery, internal carotid artery,aortic arch, femoral artery, peripheral artery, and/or ciliary artery.It is contemplated that delivery may be through or to an arteriole orcapillary.

Injection into the tumor vasculature is specifically contemplated fordiscrete, solid, accessible tumors. Local, regional or systemicadministration also may be appropriate. For tumors of >4 cm, the volumeto be administered will be about 4-10 ml (preferably 10 ml), while fortumors of <4 cm, a volume of about 1-3 ml will be used (preferably 3ml). Multiple injections delivered as single dose comprise about 0.1 toabout 0.5 ml volumes. The viral particles may advantageously becontacted by administering multiple injections to the tumor, spaced atapproximately 1 cm intervals.

In the case of surgical intervention, the present invention may be usedpreoperatively, to render an inoperable tumor subject to resection.Alternatively, the present invention may be used at the time of surgery,and/or thereafter, to treat residual or metastatic disease. For example,a resected tumor bed may be injected or perfused with a formulationcomprising a poxvirus polypeptide or a poxvirus comprising a mutationthat renders the poxvirus advantageous for treatment of cancer or cancercells. The perfusion may be continued post-resection, for example, byleaving a catheter implanted at the site of the surgery. Periodicpost-surgical treatment also is envisioned.

Continuous administration also may be applied where appropriate, forexample, where a tumor is excised and the tumor bed is treated toeliminate residual, microscopic disease. Delivery via syringe orcatherization is preferred. Such continuous perfusion may take place fora period from about 1-2 hours, to about 2-6 hours, to about 6-12 hours,to about 12-24 hours, to about 1-2 days, to about 1-2 wk or longerfollowing the initiation of treatment. Generally, the dose of thetherapeutic composition via continuous perfusion will be equivalent tothat given by a single or multiple injections, adjusted over a period oftime during which the perfusion occurs. It is further contemplated thatlimb perfusion may be used to administer therapeutic compositions of thepresent invention, particularly in the treatment of melanomas andsarcomas.

Treatment regimens may vary as well, and often depend on tumor type,tumor location, disease progression, and health and age of the patient.Obviously, certain types of tumor will require more aggressivetreatment, while at the same time, certain patients cannot tolerate moretaxing protocols. The clinician will be best suited to make suchdecisions based on the known efficacy and toxicity (if any) of thetherapeutic formulations.

In certain embodiments, the tumor being treated may not, at leastinitially, be resectable. Treatments with therapeutic viral constructsmay increase the resectability of the tumor due to shrinkage at themargins or by elimination of certain particularly invasive portions.Following treatments, resection may be possible. Additional treatmentssubsequent to resection will serve to eliminate microscopic residualdisease at the tumor site.

A typical course of treatment, for a primary tumor or a post-excisiontumor bed, will involve multiple doses. Typical primary tumor treatmentinvolves a 6 dose application over a two-week period. The two-weekregimen may be repeated one, two, three, four, five, six or more times.During a course of treatment, the need to complete the planned dosingsmay be re-evaluated.

The treatments may include various “unit doses.” Unit dose is defined ascontaining a predetermined-quantity of the therapeutic composition. Thequantity to be administered, and the particular route and formulation,are within the skill of those in the clinical arts. A unit dose need notbe administered as a single injection but may comprise continuousinfusion over a set period of time. Unit dose of the present inventionmay conveniently be described in terms of plaque forming units (pfu) fora viral construct. Unit doses range from 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸,10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³ pfu and higher. Alternatively, depending onthe kind of virus and the titer attainable, one will deliver 1 to 100,10 to 50, 100-1000, or up to about 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸,1×10⁹, 1×10¹⁰, 1×10¹¹, 1×10¹², 1×10¹³, 1×10¹⁴, or 1×10¹⁵ or higherinfectious viral particles (vp) to the patient or to the patient'scells.

B. Injectable Compositions and Formulations

The preferred method for the delivery of an expression construct orvirus encoding all or part of a poxvirus genome to cancer or tumor cellsin the present invention is via intratumoral injection. However, thepharmaceutical compositions disclosed herein may alternatively beadministered parenterally, intravenously, intradermally,intramuscularly, transdermally or even intraperitoneally as described inU.S. Pat. No. 5,543,158; U.S. Pat. No. 5,641,515 and U.S. Pat. No.5,399,363 (each specifically incorporated herein by reference in itsentirety).

Injection of nucleic acid constructs may be delivered by syringe or anyother method used for injection of a solution, as long as the expressionconstruct can pass through the particular gauge of needle required forinjection. A novel needleless injection system has recently beendescribed (U.S. Pat. No. 5,846,233) having a nozzle defining an ampulechamber for holding the solution and an energy device for pushing thesolution out of the nozzle to the site of delivery. A syringe system hasalso been described for use in gene therapy that permits multipleinjections of predetermined quantities of a solution precisely at anydepth (U.S. Pat. No. 5,846,225).

Solutions of the active compounds as free base or pharmacologicallyacceptable salts may be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions may also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms. The pharmaceutical forms suitable for injectable useinclude sterile aqueous solutions or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersions (U.S. Pat. No. 5,466,468, specifically incorporated hereinby reference in its entirety). In all cases the form must be sterile andmust be fluid to the extent that easy syringability exists. It must bestable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms, such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (e.g., glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and/or vegetable oils. Proper fluidity may bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous, intratumoral and intraperitonealadministration. In this connection, sterile aqueous media that can beemployed will be known to those of skill in the art in light of thepresent disclosure. For example, one dosage may be dissolved in 1 ml ofisotonic NaCl solution and either added to 1000 ml of hypodermoclysisfluid or injected at the proposed site of infusion, (see for example,“Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and1570-1580). Some variation in dosage will necessarily occur depending onthe condition of the subject being treated. The person responsible foradministration will, in any event, determine the appropriate dose forthe individual subject. Moreover, for human administration, preparationsshould meet sterility, pyrogenicity, general safety and purity standardsas required by FDA Office of Biologics standards.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The compositions disclosed herein may be formulated in a neutral or saltform. Pharmaceutically-acceptable salts, include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric, mandelic, andthe like. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, histidine, procaine and the like. Upon formulation,solutions will be administered in a manner compatible with the dosageformulation and in such amount as is therapeutically effective. Theformulations are easily administered in a variety of dosage forms suchas injectable solutions, drug release capsules and the like.

As used herein, “carrier” includes any and all solvents, dispersionmedia, vehicles, coatings, diluents, antibacterial and antifungalagents, isotonic and absorption delaying agents, buffers, carriersolutions, suspensions, colloids, and the like. The use of such mediaand agents for pharmaceutical active substances is well known in theart. Except insofar as any conventional media or agent is incompatiblewith the active ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions.

The phrase “pharmaceutically-acceptable” or“pharmacologically-acceptable” refers to molecular entities andcompositions that do not produce an allergic or similar untowardreaction when administered to a human. The preparation of an aqueouscomposition that contains a protein as an active ingredient is wellunderstood in the art. Typically, such compositions are prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for solution in, or suspension in, liquid prior to injectioncan also be prepared.

C. Combination Treatments

The compounds and methods of the present invention may be used in thecontext of hyperproliferative diseases/conditions including cancer andatherosclerosis. In order to increase the effectiveness of a treatmentwith the compositions of the present invention, such as attenuatedvaccinia viruses, it may be desirable to combine these compositions withother agents effective in the treatment of those diseases andconditions. For example, the treatment of a cancer may be implementedwith therapeutic compounds of the present invention and otheranti-cancer therapies, such as anti-cancer agents or surgery.

Various combinations may be employed; for example, an attenuatedpoxvirus, such as vaccinia virus, is “A” and the secondary anti-cancertherapy is “B”:

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B B/B/B/A B/B/A/BA/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/AA/A/B/A

Administration of the therapeutic expression constructs of the presentinvention to a patient will follow general protocols for theadministration of that particular secondary therapy, taking into accountthe toxicity, if any, of the poxvirus treatment. It is expected that thetreatment cycles would be repeated as necessary. It also is contemplatedthat various standard therapies, as well as surgical intervention, maybe applied in combination with the described cancer or tumor celltherapy.

1. Anti-Cancer Therapy

An “anti-cancer” agent is capable of negatively affecting cancer in asubject, for example, by killing cancer cells, inducing apoptosis incancer cells, reducing the growth rate of cancer cells, reducing theincidence or number of metastases, reducing tumor size, inhibiting tumorgrowth, reducing the blood supply to a tumor or cancer cells, promotingan immune response against cancer cells or a tumor, preventing orinhibiting the progression of cancer, or increasing the lifespan of asubject with cancer. Anti-cancer agents include biological agents(biotherapy), chemotherapy agents, and radiotherapy agents. Moregenerally, these other compositions would be provided in a combinedamount effective to kill or inhibit proliferation of the cell. Thisprocess may involve contacting the cells with the expression constructand the agent(s) or multiple factor(s) at the same time. This may beachieved by contacting the cell with a single composition orpharmacological formulation that includes both agents, or by contactingthe cell with two distinct compositions or formulations, at the sametime, wherein one composition includes the expression construct and theother includes the second agent(s).

Tumor cell resistance to chemotherapy and radiotherapy agents representsa major problem in clinical oncology. One goal of current cancerresearch is to find ways to improve the efficacy of chemo- andradiotherapy by combining it with gene therapy. For example, the herpessimplex-thymidine kinase (HS-tK) gene, when delivered to brain tumors bya retroviral vector system, successfully induced susceptibility to theantiviral agent ganciclovir (Culver et al., 1992). In the context of thepresent invention, it is contemplated that poxvirus therapy could beused similarly in conjunction with chemotherapeutic, radiotherapeutic,immunotherapeutic or other biological intervention, in addition to otherpro-apoptotic or cell cycle regulating agents.

Alternatively, the gene therapy may precede or follow the other agenttreatment by intervals ranging from minutes to weeks. In embodimentswhere the other agent and expression construct are applied separately tothe cell, one would generally ensure that a significant period of timedid not expire between the time of each delivery, such that the agentand expression construct would still be able to exert an advantageouslycombined effect on the cell. In such instances, it is contemplated thatone may contact the cell with both modalities within about 12-24 h ofeach other and, more preferably, within about 6-12 h of each other. Insome situations, it may be desirable to extend the time period fortreatment significantly, however, where several days (2, 3, 4, 5, 6 or7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between therespective administrations.

a. Chemotherapy

Cancer therapies also include a variety of combination therapies withboth chemical and radiation based treatments. Combination chemotherapiesinclude, for example, cisplatin (CDDP), carboplatin, procarbazine,mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan,chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin,doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16),tamoxifen, raloxifene, estrogen receptor binding agents, taxol,gemcitabien, navelbine, farnesyl-protein transferase inhibitors,transplatinum, 5-fluorouracil, vincristine, vinblastine andmethotrexate, Temazolomide (an aqueous form of DTIC), or any analog orderivative variant of the foregoing. The combination of chemotherapywith biological therapy is known as biochemotherapy.

b. Radiotherapy

Other factors that cause DNA damage and have been used extensivelyinclude what are commonly known as γ-rays, X-rays, and/or the directeddelivery of radioisotopes to tumor cells. Other forms of DNA damagingfactors are also contemplated such as microwaves and UV-irradiation. Itis most likely that all of these factors effect a broad range of damageon DNA, on the precursors of DNA, on the replication and repair of DNA,and on the assembly and maintenance of chromosomes. Dosage ranges forX-rays range from daily doses of 50 to 200 roentgens for prolongedperiods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens.Dosage ranges for radioisotopes vary widely, and depend on the half-lifeof the isotope, the strength and type of radiation emitted, and theuptake by the neoplastic cells.

The terms “contacted” and “exposed,” when applied to a cell, are usedherein to describe the process by which a therapeutic construct and achemotherapeutic or radiotherapeutic agent are delivered to a targetcell or are placed in direct juxtaposition with the target cell. Toachieve cell killing or stasis, both agents are delivered to a cell in acombined amount effective to kill the cell or prevent it from dividing.

c. Immunotherapy

Immunotherapeutics, generally, rely on the use of immune effector cellsand molecules to target and destroy cancer cells. The immune effectormay be, for example, an antibody specific for some marker on the surfaceof a tumor cell. The antibody alone may serve as an effector of therapyor it may recruit other cells to actually effect cell killing. Theantibody also may be conjugated to a drug or toxin (chemotherapeutic,radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) andserve merely as a targeting agent. Alternatively, the effector may be alymphocyte carrying a surface molecule that interacts, either directlyor indirectly, with a tumor cell target. Various effector cells includecytotoxic T cells and NK cells. The combination of therapeuticmodalities, i.e., direct cytotoxic activity and inhibition or reductionof certain poxvirus polypeptides would provide therapeutic benefit inthe treatment of cancer.

Immunotherapy could also be used as part of a combined therapy. Thegeneral approach for combined therapy is discussed below. In one aspectof immunotherapy, the tumor cell must bear some marker that is amenableto targeting, i.e., is not present on the majority of other cells. Manytumor markers exist and any of these may be suitable for targeting inthe context of the present invention. Common tumor markers includecarcinoembryonic antigen, prostate specific antigen, urinary tumorassociated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG,Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor, lamininreceptor, erb B and p155. An alternative aspect of immunotherapy is toanticancer effects with immune stimulatory effects Immune stimulatingmolecules also exist including: cytokines such as IL-2, IL-4, IL-12,GM-CSF, IFNγ, chemokines such as MIP-1, MCP-1, IL-8 and growth factorssuch as FLT3 ligand. Combining immune stimulating molecules, either asproteins or using gene delivery in combination with a tumor suppressorsuch as mda-7 has been shown to enhance anti-tumor effects (Ju et al.,2000).

As discussed earlier, examples of immunotherapies currently underinvestigation or in use are immune adjuvants (e.g., Mycobacterium bovis,Plasmodium falciparum, dinitrochlorobenzene and aromatic compounds)(U.S. Pat. No. 5,801,005; U.S. Pat. No. 5,739,169; Hui and Hashimoto,1998; Christodoulides et al., 1998), cytokine therapy (e.g., interferonsα, β and γ; IL-1, GM-CSF and TNF) (Bukowski et al., 1998; Davidson etal., 1998; Hellstrand et al., 1998) gene therapy (e.g., TNF, IL-1, IL-2,p53) (Qin et al., 1998; Austin-Ward and Villaseca, 1998; U.S. Pat. No.5,830,880 and U.S. Pat. No. 5,846,945) and monoclonal antibodies (e.g.,anti-ganglioside GM2, anti-HER-2, anti-p185) (Pietras et al., 1998;Hanibuchi et al., 1998; U.S. Pat. No. 5,824,311). Herceptin(trastuzumab) is a chimeric (mouse-human) monoclonal antibody thatblocks the HER2-neu receptor. It possesses anti-tumor activity and hasbeen approved for use in the treatment of malignant tumors (Dillman,1999). Combination therapy of cancer with herceptin and chemotherapy hasbeen shown to be more effective than the individual therapies. Thus, itis contemplated that one or more anti-cancer therapies may be employedwith the poxvirus-related therapies described herein.

Passive Immunotherapy.

A number of different approaches for passive immunotherapy of cancerexist. They may be broadly categorized into the following: injection ofantibodies alone; injection of antibodies coupled to toxins orchemotherapeutic agents; injection of antibodies coupled to radioactiveisotopes; injection of anti-idiotype antibodies; and finally, purging oftumor cells in bone marrow.

Preferably, human monoclonal antibodies are employed in passiveimmunotherapy, as they produce few or no side effects in the patient.However, their application is somewhat limited by their scarcity andhave so far only been administered intralesionally. Human monoclonalantibodies to ganglioside antigens have been administeredintralesionally to patients suffering from cutaneous recurrent melanoma(Irie and Morton, 1986). Regression was observed in six out of tenpatients, following, daily or weekly, intralesional injections. Inanother study, moderate success was achieved from intralesionalinjections of two human monoclonal antibodies (Irie et al., 1989).

It may be favorable to administer more than one monoclonal antibodydirected against two different antigens or even antibodies with multipleantigen specificity. Treatment protocols also may include administrationof lymphokines or other immune enhancers as described by Bajorin et al.(1988). The development of human monoclonal antibodies is described infurther detail elsewhere in the specification.

Active Immunotherapy.

In active immunotherapy, an antigenic peptide, polypeptide or protein,or an autologous or allogenic tumor cell composition or “vaccine” isadministered, generally with a distinct bacterial adjuvant (Ravindranathand Morton, 1991; Morton et al., 1992; Mitchell et al., 1990; Mitchellet al., 1993). In melanoma immunotherapy, those patients who elicit highIgM response often survive better than those who elicit no or low IgMantibodies (Morton et al., 1992). IgM antibodies are often transientantibodies and the exception to the rule appears to be anti-gangliosideor anti-carbohydrate antibodies.

Adoptive Immunotherapy.

In adoptive immunotherapy, the patient's circulating lymphocytes, ortumor infiltrated lymphocytes, are isolated in vitro, activated bylymphokines such as IL-2 or transduced with genes for tumor necrosis,and readministered (Rosenberg et al., 1988; 1989). To achieve this, onewould administer to an animal, or human patient, an immunologicallyeffective amount of activated lymphocytes in combination with anadjuvant-incorporated antigenic peptide composition as described herein.The activated lymphocytes will most preferably be the patient's owncells that were earlier isolated from a blood or tumor sample andactivated (or “expanded”) in vitro. This form of immunotherapy hasproduced several cases of regression of melanoma and renal carcinoma,but the percentage of responders were few compared to those who did notrespond.

d. Genes

In yet another embodiment, the secondary treatment is a gene therapy inwhich a therapeutic polynucleotide is administered before, after, or atthe same time as an attenuated poxvirus is administered. Delivery of apoxvirus in conjunction with a vector encoding one of the following geneproducts will have a combined anti-cancer effect on target tissues.Alternatively, the poxvirus may be engineered as a viral vector toinclude the therapeutic polynucleotide. A variety of proteins areencompassed within the invention, some of which are described below.Table 7 lists various genes that may be targeted for gene therapy ofsome form in combination with the present invention.

Inducers of Cellular Proliferation.

The proteins that induce cellular proliferation further fall intovarious categories dependent on function. The commonality of all ofthese proteins is their ability to regulate cellular proliferation. Forexample, a form of PDGF, the sis oncogene, is a secreted growth factor.Oncogenes rarely arise from genes encoding growth factors, and at thepresent, sis is the only known naturally-occurring oncogenic growthfactor. In one embodiment of the present invention, it is contemplatedthat anti-sense mRNA directed to a particular inducer of cellularproliferation is used to prevent expression of the inducer of cellularproliferation.

The proteins FMS, ErbA, ErbB and neu are growth factor receptors.Mutations to these receptors result in loss of regulatable function. Forexample, a point mutation affecting the transmembrane domain of the Neureceptor protein results in the neu oncogene. The erbA oncogene isderived from the intracellular receptor for thyroid hormone. Themodified oncogenic ErbA receptor is believed to compete with theendogenous thyroid hormone receptor, causing uncontrolled growth.

The largest class of oncogenes includes the signal transducing proteins(e.g., Src, Abl and Ras). The protein Src is a cytoplasmicprotein-tyrosine kinase, and its transformation from proto-oncogene tooncogene in some cases, results via mutations at tyrosine residue 527.In contrast, transformation of GTPase protein ras from proto-oncogene tooncogene, in one example, results from a valine to glycine mutation atamino acid 12 in the sequence, reducing ras GTPase activity.

The proteins Jun, Fos and Myc are proteins that directly exert theireffects on nuclear functions as transcription factors.

Inhibitors of Cellular Proliferation.

The tumor suppressor oncogenes function to inhibit excessive cellularproliferation. The inactivation of these genes destroys their inhibitoryactivity, resulting in unregulated proliferation. The tumor suppressorsp53, p16 and C-CAM are described below.

In addition to p53, which has been described above, another inhibitor ofcellular proliferation is p16. The major transitions of the eukaryoticcell cycle are triggered by cyclin-dependent kinases, or CDK's. One CDK,cyclin-dependent kinase 4 (CDK4), regulates progression through the G₁.The activity of this enzyme may be to phosphorylate Rb at late G₁. Theactivity of CDK4 is controlled by an activating subunit, D-type cyclin,and by an inhibitory subunit, the p16^(INK4) has been biochemicallycharacterized as a protein that specifically binds to and inhibits CDK4,and thus may regulate Rb phosphorylation (Serrano et al., 1993; Serranoet al., 1995). Since the p16^(INK4) protein is a CDK4 inhibitor(Serrano, 1993), deletion of this gene may increase the activity ofCDK4, resulting in hyperphosphorylation of the Rb protein. p16 also isknown to regulate the function of CDK6.

p16^(INK4) belongs to a newly described class of CDK-inhibitory proteinsthat also includes p16^(B), p19, p21^(WAF1), and p27^(KIP1). Thep16^(INK4) gene maps to 9p21, a chromosome region frequently deleted inmany tumor types. Homozygous deletions and mutations of the p16^(INK4)gene are frequent in human tumor cell lines. This evidence suggests thatthe p16^(INK4) gene is a tumor suppressor gene. This interpretation hasbeen challenged, however, by the observation that the frequency of thep16^(INK4) gene alterations is much lower in primary uncultured tumorsthan in cultured cell lines (Caldas et al., 1994; Cheng et al., 1994;Hussussian et al., 1994; Kamb et al., 1994; Kamb et al., 1994; Mon etal., 1994; Okamoto et al., 1994; Nobori et al., 1994; Orlow et al.,1994; Arap et al., 1995). Restoration of wild-type p16^(INK4) functionby transfection with a plasmid expression vector reduced colonyformation by some human cancer cell lines (Okamoto, 1994; Arap, 1995).

Other genes that may be employed according to the present inventioninclude Rb, APC, DCC, NF-1, NF-2, WT-1, MEN-I, MEN-II, zac1, p73, VHL,MMAC1/PTEN, DBCCR-1, FCC, rsk-3, p27, p27/p16 fusions, p21/p27 fusions,anti-thrombotic genes (e.g., COX-1, TFPI), PGS, Dp, E2F, ras, myc, nett,raf, erb, fins, trk, ret, gsp, hst, abl, E1A, p300, genes involved inangiogenesis (e.g., VEGF, FGF, thrombospondin, BAI-1, GDAIF, or theirreceptors) and MCC.

Regulators of Programmed Cell Death.

Apoptosis, or programmed cell death, is an essential process for normalembryonic development, maintaining homeostasis in adult tissues, andsuppressing carcinogenesis (Kerr et al., 1972). The Bcl-2 family ofproteins and ICE-like proteases have been demonstrated to be importantregulators and effectors of apoptosis in other systems. The Bcl-2protein, discovered in association with follicular lymphoma, plays aprominent role in controlling apoptosis and enhancing cell survival inresponse to diverse apoptotic stimuli (Bakhshi et al., 1985; Cleary andSklar, 1985; Cleary et al., 1986; Tsujimoto et al., 1985; Tsujimoto andCroce, 1986). The evolutionarily conserved Bcl-2 protein now isrecognized to be a member of a family of related proteins, which can becategorized as death agonists or death antagonists.

Subsequent to its discovery, it was shown that Bcl-2 acts to suppresscell death triggered by a variety of stimuli. Also, it now is apparentthat there is a family of Bcl-2 cell death regulatory proteins whichshare in common structural and sequence homologies. These differentfamily members have been shown to either possess similar functions toBcl-2 (e.g., Bcl_(XL), Bcl_(W), Bcl_(S), Mcl-1, A1, Bfl-1) or counteractBcl-2 function and promote cell death (e.g., Bax, Bak, Bik, Bim, Bid,Bad, Harakiri).

D. Surgery

Approximately 60% of persons with cancer will undergo surgery of sometype, which includes preventative, diagnostic or staging, curative andpalliative surgery. Curative surgery is a cancer treatment that may beused in conjunction with other therapies, such as the treatment of thepresent invention, chemotherapy, radiotherapy, hormonal therapy, genetherapy, immunotherapy and/or alternative therapies.

Curative surgery includes resection in which all or part of canceroustissue is physically removed, excised, and/or destroyed. Tumor resectionrefers to physical removal of at least part of a tumor. In addition totumor resection, treatment by surgery includes laser surgery,cryosurgery, electrosurgery, and microscopically controlled surgery(Mohs' surgery). It is further contemplated that the present inventionmay be used in conjunction with removal of superficial cancers,precancers, or incidental amounts of normal tissue.

Upon excision of part of all of cancerous cells, tissue, or tumor, acavity may be formed in the body. Treatment may be accomplished byperfusion, direct injection or local application of the area with anadditional anti-cancer therapy. Such treatment may be repeated, forexample, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. Thesetreatments may be of varying dosages as well.

E. Other Agents

It is contemplated that other agents may be used in combination with thepresent invention to improve the therapeutic efficacy of treatment.These additional agents include immunomodulatory agents, agents thataffect the upregulation of cell surface receptors and GAP junctions,cytostatic and differentiation agents, inhibitors of cell adhesion,agents that increase the sensitivity of the hyperproliferative cells toapoptotic inducers, or other biological agents. Immunomodulatory agentsinclude tumor necrosis factor; interferon α, β, and γ; IL-2 and othercytokines; F42K and other cytokine analogs; or MIP-1, MIP-1β, MCP-1,RANTES, and other chemokines. It is further contemplated that theupregulation of cell surface receptors or their ligands such as Fas/Fasligand, DR4 or DR5/TRAIL (Apo-2 ligand) would potentiate the apoptoticinducing abilities of the present invention by establishment of anautocrine or paracrine effect on hyperproliferative cells. Increasesintercellular signaling by elevating the number of GAP junctions wouldincrease the anti-hyperproliferative effects on the neighboringhyperproliferative cell population. In other embodiments, cytostatic ordifferentiation agents can be used in combination with the presentinvention to improve the anti-hyperproliferative efficacy of thetreatments Inhibitors of cell adhesion are contemplated to improve theefficacy of the present invention. Examples of cell adhesion inhibitorsare focal adhesion kinase (FAKs) inhibitors and Lovastatin. It isfurther contemplated that other agents that increase the sensitivity ofa hyperproliferative cell to apoptosis, such as the antibody c225, couldbe used in combination with the present invention to improve thetreatment efficacy.

Apo2 ligand (Apo2L, also called TRAIL) is a member of the tumor necrosisfactor (TNF) cytokine family. TRAIL activates rapid apoptosis in manytypes of cancer cells, yet is not toxic to normal cells. TRAIL mRNAoccurs in a wide variety of tissues. Most normal cells appear to beresistant to TRAIL's cytotoxic action, suggesting the existence ofmechanisms that can protect against apoptosis induction by TRAIL. Thefirst receptor described for TRAIL, called death receptor 4 (DR4),contains a cytoplasmic “death domain”; DR4 transmits the apoptosissignal carried by TRAIL. Additional receptors have been identified thatbind to TRAIL. One receptor, called DR5, contains a cytoplasmic deathdomain and signals apoptosis much like DR4. The DR4 and DR5 mRNAs areexpressed in many normal tissues and tumor cell lines. Recently, decoyreceptors such as DcR1 and DcR2 have been identified that prevent TRAILfrom inducing apoptosis through DR4 and DR5. These decoy receptors thusrepresent a novel mechanism for regulating sensitivity to apro-apoptotic cytokine directly at the cell's surface. The preferentialexpression of these inhibitory receptors in normal tissues suggests thatTRAIL may be useful as an anticancer agent that induces apoptosis incancer cells while sparing normal cells. (Marsters et al., 1999).

There have been many advances in the therapy of cancer following theintroduction of cytotoxic chemotherapeutic drugs. However, one of theconsequences of chemotherapy is the development/acquisition ofdrug-resistant phenotypes and the development of multiple drugresistance. The development of drug resistance remains a major obstaclein the treatment of such tumors and therefore, there is an obvious needfor alternative approaches such as gene therapy.

Another form of therapy for use in conjunction with chemotherapy,radiation therapy or biological therapy includes hyperthermia, which isa procedure in which a patient's tissue is exposed to high temperatures(up to 106° F.). External or internal heating devices may be involved inthe application of local, regional, or whole-body hyperthermia. Localhyperthermia involves the application of heat to a small area, such as atumor. Heat may be generated externally with high-frequency wavestargeting a tumor from a device outside the body. Internal heat mayinvolve a sterile probe, including thin, heated wires or hollow tubesfilled with warm water, implanted microwave antennae, or radiofrequencyelectrodes.

A patient's organ or a limb is heated for regional therapy, which isaccomplished using devices that produce high energy, such as magnets.Alternatively, some of the patient's blood may be removed and heatedbefore being perfused into an area that will be internally heated.Whole-body heating may also be implemented in cases where cancer hasspread throughout the body. Warm-water blankets, hot wax, inductivecoils, and thermal chambers may be used for this purpose.

Hormonal therapy may also be used in conjunction with the presentinvention or in combination with any other cancer therapy previouslydescribed. The use of hormones may be employed in the treatment ofcertain cancers such as breast, prostate, ovarian, or cervical cancer tolower the level or block the effects of certain hormones such astestosterone or estrogen. This treatment is often used in combinationwith at least one other cancer therapy as a treatment option or toreduce the risk of metastases.

TABLE 6 Oncogenes Gene Source Human Disease Function Growth FactorsHST/KS Transfection FGF family member INT-2 MMTV promoter FGF familymember Insertion INTI/WNTI MMTV promoter Factor-like Insertion SISSimian sarcoma virus PDGF B Receptor Tyrosine Kinases ERBB/HER Avianerythroblastosis Amplified, deleted EGF/TGF-α/ virus; ALV promoterSquamous cell Amphiregulin/ insertion; amplified Cancer; glioblastomaHetacellulin receptor human tumors ERBB-2/NEU/HER-2 Transfected from ratAmplified breast, Regulated by NDF/ Glioblastomas Ovarian, gastricHeregulin and EGF- cancers Related factors FMS SM feline sarcoma virusCSF-1 receptor KIT HZ feline sarcoma virus MGF/Steel receptorHematopoieis TRK Transfection from NGF (nerve growth human colon cancerFactor) receptor MET Transfection from Scatter factor/HGF humanosteosarcoma Receptor RET Translocations and point Sporadic thyroidcancer; Orphan receptor Tyr mutations Familial medullary Kinase thyroidcancer; multiple endocrine neoplasias 2A and 2B ROS URII avian sarcomaOrphan receptor Tyr Virus Kinase PDGF receptor Translocation ChronicTEL(ETS-like Myelomonocytic Transcription factor)/ Leukemia PDGFreceptor gene Fusion TGF-β receptor Colon carcinoma Mismatch mutationtarget NONRECEPTOR TYROSINE KINASES ABI. Abelson Mul.V Chronicmyelogenous Interact with RB, RNA Leukemia translocation Polymerase,CRK, with BCR CBL FPS/FES Avian Fujinami SV; GA FeSV LCK Mul.V (murineleukemia Src family; T cell virus) promoter Signaling; interactsinsertion CD4/CD8 T cells SRC Avian Rous sarcoma Membrane-associatedVirus Tyr kinase with signaling function; activated by receptor kinasesYES Avian Y73 virus Src family; signaling SER/THR PROTEIN KINASES AKTAKT8 murine retrovirus Regulated by PI(3)K?; regulate 70-kd S6 k? MOSMaloney murine SV GVBD; cystostatic factor; MAP kinase kinase PIM-1Promoter insertion Mouse RAF/MIL 3611 murine SV; MH2 Signaling in RASavian SV Pathway MISCELLANEOUS CELL SURFACE APC Tumor suppressor Coloncancer Interacts with catenins DCC Tumor suppressor Colon cancer CAMdomains E-cadherin Candidate tumor Breast cancer Extracellular homotypicSuppressor binding; intracellular interacts with catenins PTC/NBCCSTumor suppressor and Nevoid basal cell cancer 12 transmembraneDrosophila homology Syndrome (Gorline domain; signals syndrome) throughGli homogue CI to antagonize Hedgehog pathway TAN-1 Notch TranslocationT-ALI. Signaling homologue MISCELLANEOUS SIGNALING BCL-2 TranslocationB-cell lymphoma Apoptosis CBL Mu Cas NS-1 V Tyrosine- PhosphorylatedRING finger interact Abl CRK CT1010 ASV Adapted SH2/SH3 interact AblDPC4 Tumor suppressor Pancreatic cancer TGF-β-related signaling PathwayMAS Transfection and Possible angiotensin Tumorigenicity Receptor NCKAdaptor SH2/SH3 GUANINE NUCLEOTIDE EXCHANGERS AND BINDING PROTEINS BCRTranslocated with ABL Exchanger; protein in CML Kinase DBL TransfectionExchanger GSP NF-1 Hereditary tumor Tumor suppressor RAS GAP SuppressorNeurofibromatosis OST Transfection Exchanger Harvey-Kirsten, N- HaRatSV; Ki RaSV; Point mutations in many Signal cascade RAS Balb-MoMuSV;human tumors Transfection VAV Transfection S112/S113; exchanger NUCLEARPROTEINS AND TRANSCRIPTION FACTORS BRCA1 Heritable suppressor MammaryLocalization unsettled Cancer/ovarian cancer BRCA2 Heritable suppressorMammary cancer Function unknown ERBA Avian erythroblastosis Thyroidhormone Virus receptor (transcription) ETS Avian E26 virus DNA bindingEVII MuLV promotor AML Transcription factor Insertion FOS FBI/FBR murineTranscription factor osteosarcoma viruses with c-JUN GLI Amplifiedglioma Glioma Zinc finger; cubitus Interruptus homologue is in hedgehogsignaling pathway; inhibitory link PTC and hedgehog HMGI/LIMTranslocation t(3:12) Lipoma Gene fusions high t(12:15) mobility groupHMGI-C (XT-hook) and transcription factor LIM or acidic domain JUNASV-17 Transcription factor AP-1 with FOS MLL/VHRX +Translocation/fusion Acute myeloid leukemia Gene fusion of DNA- ELI/MENELL with MLL binding and methyl Trithorax-like gene transferase MLL withELI RNA pol II Elongation factor MYB Avian myeloblastosis DNA bindingVirus MYC Avian MC29; Burkitt's lymphoma DNA binding with TranslocationB-cell MAX partner; cyclin Lymphomas; promoter Regulation; interactInsertion avian RB?; regulate leukosis Apoptosis? Virus N-MYC AmplifiedNeuroblastoma L-MYC Lung cancer REL Avian NF-κB familyRetriculoendotheliosis Transcription factor Virus SKI Avian SKV770Transcription factor Retrovirus VHL Heritable suppressor VonHippel-Landau Negative regulator or Syndrome elongin; transcriptionalelongation complex WT-1 Wilm's tumor Transcription factor CELL CYCLE/DNADAMAGE RESPONSE ATM Hereditary disorder Ataxia-telangiectasiaProtein/lipid kinase Homology; DNA damage response upstream in P53pathway BCL-2 Translocation Follicular lymphoma Apoptosis FACC Pointmutation Fanconi's anemia group C (predisposition Leukemia FHIT Fragilesite 3p14.2 Lung carcinoma Histidine triad-related Diadenosine 5′,3″″-P¹.p⁴ tetraphosphate Asymmetric hydrolase hMLI/MutL HNPCC Mismatchrepair; MutL Homologue HMSH2/MutS HNPCC Mismatch repair; MutS HomologueHPMS1 HNPCC Mismatch repair; MutL Homologue hPMS2 HNPCC Mismatch repair;MutL Homologue INK4/MTS1 Adjacent INK-4B at Candidate MTS1 p16 CDKinhibitor 9p21; CDK complexes Suppressor and MLM melanoma geneINK4B/MTS2 Candidate suppressor p15 CDK inhibitor MDM-2 AmplifiedSarcoma Negative regulator p53 p53 Association with SV40 Mutated >50%human Transcription factor; T antigen Tumors, including Checkpointcontrol; hereditary Li-Fraumeni apoptosis syndrome PRAD1/BCL1Translocation with Parathyroid adenoma; Cyclin D Parathyroid hormoneB-CLL or IgG RB Hereditary Retinoblastoma; Interact cyclin/cdk;Retinoblastoma; Osteosarcoma; breast regulate E2F Association with manyCancer; other sporadic transcription factor DNA virus tumor CancersAntigens XPA Xeroderma Excision repair; photo- Pigmentosum; skin productrecognition; Cancer predisposition zinc finger

VIII. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1: Material and Methods

Viruses and Cell Lines.

The panel of wild type poxvirus strains (Wyeth, Western Reserve (WR),USSR, Tian Tan, Tash Kent, Patwadangar, Lister, King, IHD-W, IHD-J andEvans) was kindly provided by Dr Geoff Smith, Imperial College, London.Human Adenovirus serotype 5 (Ad5) was obtained from ATCC. The Viralgrowth factor (VGF) deleted strain of WR (vSC20) was kindly provided byDr Bernie Moss, NIH. The thymidine kinase deleted strain of WR (vJS6)and the TK-, VGF-double deleted strain of WR (vvDD) are described inPuhlmann et al. (2000) and McCart et al. (2001). WR strain expressingfirefly luciferase was kindly provided by Dr Gary Luker, (Uni Michigan).

Vaccinia strain JX-963 was constructed by recombination of a version ofthe pSC65 plasmid containing the E. coli gpt and human GM-CSF genes(under the control of the p7.5 and pSE/L promoters respectively) intothe thymidine kinase gene of the vSC20 (VGF deleted) strain of WR.Further selection of white plaques after propagation of the virus inX-Gal produced a virus with non-functioning lacZ (lacZ is expressed fromwithin VGF in vSC20). Correct insertion into the TK gene and loss oflacZ function was verified by sequencing and GM-CSF production verifiedby ELISA.

The vvDD expressing luciferase was constructed by insertion of a versionof the pSC65 plasmid with luciferase under control of the p7.5 promoterinto vSC20. Bioluminescence was verified using an IVIS 50 system(Xenogen, Alameda).

The human tumor cell lines include A2780 (Ovarian, obtained from ECACC),A549 (lung, obtained from ECACC), HCT 116, HT-29 and SW620 (colon,obtained from ATCC), HT-1080 (fibrosarcoma, obtained from ATCC), LNCaP(prostate, obtained from ATCC), PANC-1 (pancreatic, obtained from ATCC),MCF-7 (breast, obtained from ATCC). Non-transformed cells include MRC-5(lung fibroblast, obtained from ATCC), Beas-2B (bronchial epithelial,kindly provided by Tony Reid, UCSD) and the primary, normal cells NHBE(Normal human bronchial epithelial) and SAEC (Small airway bronchialepithelial), both obtained from Clonetics (Walkersville, Md.).

The mouse tumor cell lines include CMT 64 (C57/B6 lung, obtained fromCancer Research UK), JC (BALB/c mammary, obtained from ATCC), MC38(C57/B6 colon, obtained from NIH) and TIB-75 (BNL 1ME A.7R.1)(BALB/chepatic, obtained from ATCC). The cell lines NIH 3T3 and NIH 3T3overexpressing H-Ras were kindly provided by Richard Marais (ICR,London). The rabbit tumor cell line VX2 has been described previously(Kidd, 1940; Tjernberg, 1962; Chen et al., 2004).

In Vitro Replication and Cytopathic Effect Assays.

Cell lines are seeded into 6-well plates at 5×10⁶ cells/well and leftovernight. Virus was then added at a multiplicity of infection (MOI) of1.0 Plaque forming units (PFU)/cell and allowed to infect for 2 h. Atthe end of the infection the media was changed and plates incubated for48 h, the cells were then scraped into the media and collected. Cellswere lysed by three rounds of freezing and thawing followed bysonication before serial dilutions of the crude viral lysate was addedto BSC-1 cells to titer the virus. Plaque assay was performed asdescribed previously (Earl et al., 1998). Adenovirus was titered on A549cells (Earl et al., 1998). Studies are typically run in triplicate.

In order to assess the cytopathic effect (CPE) of the virus, cells wereseeded at 1000 cells/well in 96-well plates and allowed to attachovernight. Serial dilutions of the viruses to be tested were then addedto the plates in triplicate (MOI range from 100 to 0.001) and the platesincubated for a further 72 h. After this time media was replaced withmedia without serum and MTS (Promega) added to the plates. After 2-4 hincubation the absorbance at 450 nm was read on an ELISA plate reader.Cytopathic effect was determined as reduction in viability of a testwell relative to both untreated wells containing cells only (100%viable) and cell-free wells (0% viable). Results were represented as theMOI at which 50% of the cell layer was viable (effective concentration50%, EC50).

Mouse Syngeneic and Xenograft Tumor Model Studies.

Immunocompetent mice are implanted subcutaneously with syngeneic tumorcells (1×10⁶ cells/mouse), such that JC and TIB-75 cells are implantedinto BALB/c mice and MC38 and CMT 64 cells are implanted into C57/B6mice. Certain human xenograft models involve 1×10⁷ HT29 cells implantedsubcutaneously into SCID mice (all mice are aged 8-10 weeks and sexmatched). Once tumors reached 50-100 mm³ animals are regrouped andtreated as indicated. Tumor sizes were followed by caliper measurement.

Mice treated with luciferase expressing virus can be imaged using anIVIS 100 system (Xenogen, Alameda). Mice are injected intraperitoneallywith luciferin (30 mg/kg) and anesthetized (2% isoflurane) prior toimaging.

Some mice are sacrificed at times indicated post-treatment and organsare recovered for viral biodistribution or immunohistochemical studies.For viral biodistribution, organs are snap frozen and ground beforeplaque assays are performed as described. For immunohistochemistrystudies, organs are fixed in formalin before embedding in paraffinblocks for sectioning. Sections are stained with hematoxylin and eosin(H & E) and with viral coat proteins (polyclonal anti-vaccinia antibodyor polyclonal antihexon antibody for adenovirus treated animals).

Rabbit Model.

The implantation of VX2 tumors into the livers of New Zealand Whiterabbits and the measurement of tumor progression and metastasis to thelungs by CT and ultrasound scans has been described previously (Paeng etal., 2003).

Cytotoxic T-Lymphocyte (CTL) Assay.

This is performed by mixing labeled peripheral blood lymphocytes (PBLs)obtained from rabbits treated as indicated with VX2 tumor cells. After a4 h period cell apoptosis was measured by propidium iodide staining andflow cytometry.

Neutralizing Antibody Assay.

Production of anti-vaccinia neutralizing antibody is measured in theplasma obtained from rabbits post-treatment. Dilutions of plasma aremixed with 1000 PFU of vaccinia overnight before addition to a 96-wellplate containing A2780 cells. After 72 h cell viability is measured byMTS assay. Viral neutralization is measured as the dilution of plasmarequired to prevent viral inactivation.

Statistical Analyses.

Kaplan-Meier curves are compared using the Generalized Wilcoxin test.Tumor response rates and metastasis-free rates are typically comparedwith Fisher's exact test.

Example 2: Rat Tumor Model

Rats (Sprague-Dawley, Males) were exposed to carcinogen(N-Nitrosomorpholine, NNM) in their drinking water (175 mg/L) for aperiod of 8 weeks, during which time liver cirrhosis developed, followedby in situ development of tumors (hepatocellular carcinoma orcholangiocarcinoma) within the liver between weeks 16-20 on average(model previously described in Oh et al., 2002). Tumor detection andevaluation was performed by an experienced ultrasonographer usingultrasound imaging. Tumor sizes were approximately 0.75-1.5 cm. indiameter at baseline immediately prior to treatment initiation; tumorvolumes were not significantly different at baseline between the controland treatment groups (estimated mean volumes were 400-500 mm³). Controlanimals (n=17) received no treatment, whereas treated animals (n=6)received intravenous injections (via tail vein) with a poxvirus (Wyethstrain; thymidine kinase gene deletion present) expressing human GM-CSFfrom a synthetic early-late promoter (virus construct described inMastrangelo et al., 1999). Virus was administered at a dose of 10⁸plaque-forming units (titered as in Earl et al., 1998) in a total volumeof 0.75 ml; (virus suspension mixed with 10 mM Tris up to the desiredvolume) intravenously by tail vein over 60 seconds. Treatment wasrepeated every two weeks for three total doses (day 1, 15 and 29).

Over ten weeks following the initiation of treatment, the control tumorsincreased in size significantly until reaching a mean of approximately3000 mm³ (S.E. 500) (FIG. 1). Control animals needed to be sacrificedfor ethical reasons due to tumor progression at this time. All tumorshad increased in size significantly. In contrast, five of the sixtreated tumors regressed completely (below the limit of detection byultrasound). The mean tumor volume in the treated group wasapproximately 50 mm³ (S.E., <10; p<0.01 vs. controls).

Example 3: Rabbit VX2 Tumor Model

A study was performed in a VX2 rabbit carcinoma model (as described inPaeng et al., 2003). Rabbit was selected as a species because humanGM-CSF was previously demonstrated to have significant biologicalactivity in rabbits (in contrast to mice). VX2 tumors were grown inmuscle of New Zealand white rabbits and cells from a 1-2 mm³ fragment oftumor were dissociated, resuspended in 0.1 ml normal saline and wereinjected beneath the liver capsule (21 gauge needle; injection sitecovered with surgical patch with a purse-string tie) and allowed to growfor 14 days until primary tumors were established (mean diameter,1.5-2.0 cm; est. volume 2-4 cm³). VX2 cells were demonstrated to beinfectable by vaccinia poxvirus ex vivo in a standard burst assay. Tumorsizes were monitored over time by CT scanning and by ultrasound. Overthe following seven weeks, control (untreated) animals (n=18) developedtumor progression within the liver, with estimated mean tumor volumesreaching approximately 100 cm³ (S.E. approximately 20). In addition,numerous tumor metastases progressed and became detectable within thelungs and livers over time (FIGS. 2A-B). By week 7, control animals allhad detectable metastases, with a mean number of lung metastases of 17(S.E. 2.3). The median survival of these control animals was 55 days(post-treatment initiation in treated animals), and all were dead within80 days.

Treated animals (n=3) in the first experiment received a singleintravenous injection (via tail vein) with a poxvirus (Wyeth strain;thymidine kinase gene deletion present) expressing human GM-CSF from asynthetic early-late promoter (virus construct described in Mastrangeloet al., 1999). Virus was administered at a dose of 10⁹ plaque-formingunits (titered as in Earl et al., 1998) in a total volume of 7 ml;(virus suspension mixed with 10 mM Tris up to the desired volume)intravenously by ear vein over 60 seconds. By week seven, in contrast tocontrols, treated animals had no lung metastases detectable by CTscanning (FIGS. 2A-2B). Survival was significantly increased, also. By110 days post-treatment initiation, the median survival had not beenreached, and approximately 70% were still alive.

Treated animals (n=6 per group) in a second experiment received threeweekly intravenous injections (via tail vein) with either (i) JX-594, apoxvirus (Wyeth strain; thymidine kinase gene deletion present)expressing human QM-CSF from a synthetic early-late promoter (virusconstruct described in Mastrangelo et al., 1999, which is herebyincorporated by reference), (ii) vvDD, a vaccinia WR strain withdeletions in thymidine kinase and vaccinia growth factor genes (vvDD asdescribed by MeCart et al., 1999), or (iii) JX-963, vaccinia WR strainwith deletions in thymidine kinase and vaccinia growth factor genes andexpressing human GM-CSF from a synthetic early-late promoter. Virus wasadministered at a dose of 10⁸ plaque-forming units (titered as in Earlet al., 1998) in a total volume of 7 ml; (virus suspension mixed with 10nM Tris up to the desired volume) intravenously by ear vein over 60seconds. By week seven, in contrast to controls, JX-963 treated animalshad no lung metastases detectable by CT scanning (p<0.01 vs. controls)(FIG. 4). JX-594-treated animals had a mean of 8 lung tumors (S.E. 2;p<0.05 vs controls), vvDD-treated animals had a mean of 5 lung tumors(S.E. 2; p<0.05 vs controls). Of note, JX-963 and vvDD also hadsignificant efficacy against the primary tumor growth in the liver, incontrast to JX-594 at this dose (FIG. 3) and JX-963 dramaticallyincreased the survival of these animals (FIG. 5).

The GM-CSF-expressing virus JX-963 had significantly better efficacyagainst both primary tumors and lung metastases than itsnon-GM-CSF-expressing control vvDD; 2) the GM-CSF-expressing virusJX-963 had significantly better efficacy against both primary tumors andlung metastases than its GM-CSF-expressing Wyeth strain control (despitean additional deletion in the vgf gene not present in JX-594).Therefore, intravenous administration with a vaccinia expressing humanGM-CSF resulted in significantly better efficacy over the same vacciniawithout GM-CSF, and intravascular administration of a WR strain deletionmutant expressing human GM-CSF was significantly better than a Wyethstrain (standard vaccine strain) deletion mutant expressing GM-CSF.

Example 4: Systemic Cancer Efficacy with JX-963

Targeted therapies hold great promise for the treatment of cancer, butnovel agents are still needed as resistance frequently develops throughmutation of the target molecules and/or tumor escape through pathwayredundancies. Oncolytic viruses are viruses that have their replicationrestricted to malignant cell types, either inherently or through geneticengineering (Thorne et al., 2005)¹. Selective intratumoral replicationleads to virus multiplication, killing of the infected cancer cell byunique and apoptosis-independent mechanisms (oncolysis) and spread ofthe virus to other tumor cells. Virotherapeutics therefore have thepotential to effectively treat refractory cancers and clinicalproof-of-concept has been achieved with local or regional administrationfor several oncolytic viruses (Parato et al., 2005)². However, foroncolytic viruses to have a major impact on patient survival, systemicefficacy and intravenous delivery will be needed.

The inventor has therefore undertaken a stepwise design and developmentstrategy to create a more effective systemic agent. First, the inventoridentified poxviruses such as vaccinia as a virus species that hasevolved for systemic dissemination and resistance to clearance bycomplement and antibodies (Smith et al., 1997; Buller and Palumbo,1991). Vaccinia has well-defined mechanisms to allow for transport inthe blood without inactivation and can spread rapidly within tissues, italso has a long history of human use during the smallpox eradicationcampaign. A panel of vaccinia viruses used during the vaccinationprogram, and some related strains were screened for their ability toreplicate in normal (NHBE) and tumor (A2780) cells. All vaccinia strainsreplicated to higher levels in the tumor cell line than in the normalcells (FIG. 6A), but the therapeutic index (tumor to normal cellreplication ratio) varied between strains. Strains used extensively inthe laboratory (such as Western Reserve (WR)) tended to display greaterinherent tumor selectivity in vitro than their parental vaccine strains(Wyeth). This is the first time that wild type vaccinia strains havebeen shown to display inherent superior replication in tumor cell linesrelative to normal cells. This is not true for all viruses however, asAdenovirus serotype 5 (Ad5) (the backbone for the majority of oncolyticviruses in the clinic) did not display such selectivity (FIG. 10A).

Another desirable attribute for an oncolytic agent is rapid intratumoralspread (Wein et al., 2003). This can be achieved through a shortreplication cycle and early release of virus from infected cells. Theability of the WR strain of vaccinia to destroy tumor cells wastherefore examined at early time points (72 h) after infection andcompared to Ad5 and the oncolytic adenovirus strain d11520 (ONYX-015)(Heise et al., 1997) (FIG. 6B). WR displayed up to 5-logs of increasedkilling potential in tumor cells at this time relative to both Ad5 anddl1520, as well as greater tumor selectivity than either adenoviralstrain.

The major limitation of most oncolytic viruses tested to date is aninability to efficiently infect tumors following systemic delivery, asseen when 1×10⁹ plaque forming units (PFU) of Ad5 were deliveredintravenously to subcutaneous tumor models in mice (FIG. 6C and FIG.10B); this equates to a dose of 3.5×10¹² PFU in a 70 kg human, higherthan ever given to a patient. Little or no replicating virus was evidentin tumors (as detected by immunohistochemical staining for viral coatproteins 48 and 72 h after viral delivery). Vaccinia strain WR howevercould effectively traffic to and infect the tumors in these same models,with up to 50% of the tumor cells staining positive within 48 h oftreatment. Furthermore, vaccinia was able to persist in the tumor for atleast 10 days (FIG. 10B), despite the fact an immune response would havebeen initiated by this time.

In order to maximize safety, particularly for intravenous administrationin immunodeficient cancer patients, attenuating and tumor-targetinggenetic deletions were introduced into the virus. The inventor haspreviously described preferential tumor-expression of viral genes withinsertions into the vaccinia thymidine kinase (TK) gene and of TK andviral growth factor (VGF) double deletions (Puhlmann et al., 1999;McCart et al., 2001). Although the targeting mechanisms of thesedeletions were not previously demonstrated, the rationale was torestrict virus replication and oncolysis to cancer cells with elevatedE2F levels (as E2F drives production of the cellular thymidine kinasegene product (Hengstschlager et al. 1994)) and activation of theepidermal growth factor (EGF) receptor pathway (as activation of thispathway by VGF is necessary for efficient viral replication (Andrade etal., 2004)). Here it is shown that the TK and VGF double deleted virus(vvDD) displayed an impressive ability to destroy a wide range of tumorcells of different origins (FIG. 7). It was also found that singledeletions in either the vaccinia TK or the VGF genes attenuated theability of vaccinia to replicate in non-proliferating, non-transformedhuman cell lines, while the double deleted virus (vvDD) was furtherattenuated (FIG. 11). None of these strains were attenuated in theirability to replicate in human tumor cells.

It was further found that the block in the ability of the VGF-deletedvirus to replicate in non-proliferating, non-transformed cells could beovercome in cells expressing activated H-ras (FIG. 8A). It was foundthat H-ras activation led to increased replication of even WR(p=0.0094), and that VGF deletion did not inhibit viral replication inH-ras activated cells, whereas the TK deletion did (p=0.016). Thisindicates that the tumor selectivity introduced by the gene deletions invvDD is more than a simple preference for proliferating cells, sinceslowly proliferating or even non-proliferating cells could be targetedif they contained mutations in the EGF-R/Ras/MAP Kinase signalingpathway.

In order to determine whether the double deleted vaccinia (vvDD) mightproduce toxicity by targeting normal proliferating cells (such as gutepithelial, bone marrow or ovarian cells), in vivo viral gene expressionwas studied by non-invasive bioluminescence imaging (FIG. 8B) and viralbiodistribution was examined post mortem (FIG. 12). Bioluminescenceimaging following IV delivery of 1×10⁷ PFU of WR or vvDD expressingluciferase showed that both viruses displayed similar initial infectionand viral gene expression patterns (including spleen, lung, liver andtumor) (FIG. 8B). However, the bioluminescent signal from vvDD wasrapidly cleared from most organs other than the tumor, even inimmunodeficient mice, while WR continued to replicate in the targetorgans and spread to other tissues, including bone marrow, skin andbrain (FIGS. 8B and 8C). Although vvDD did produce some points ofinfection outside of the tumor, these appeared transiently and late,indicating secondary spread without replication (data not shown).Recovery of infectious viral units from tissues of mice treated IV with1×10⁹ PFU of vvDD (a lethal dose for WR) revealed that by day 8 aftertreatment the tumor displayed increasing viral titer, with over1,000-fold more viral copies per mg tissue than any other organ, whileall normal tissues were below the limits of detection or showed fallingviral titers (FIG. 12).

The anti-tumor effects of vvDD were then analyzed in immunocompetentmouse models. vvDD had significantly greater anti-tumor effects than aWyeth TK deleted vaccinia strain (the most common vaccinia strain inclinical trials, usually used as a vaccine) when both were deliveredintravenously (FIG. 13). Further studies showed that 1×10⁹ PFU of vvDDwas capable of significant anti-tumor effects when delivered by eithersystemic or intratumoral injection to both immunodeficient mice carryinghuman tumor xenografts and immunocompetent mice bearing syngeneic tumors(FIG. 13).

In order to increase the anti-tumor potential of vvDD, and to suppressthe outgrowth of microscopic tumor deposits that are not vascularized atthe time of IV dosing, the cytokine GM-CSF was inserted into the site ofthe TK gene (under the control of the synthetic E/L promoter); thisvirus was designated JX-963. Because human GM-CSF is not active inrodents but is active in rabbits (Cody et al., 2005), and in order toassess the activity against much larger primary tumors that reproduciblymetastasize, JX-963 was used in a rabbit model with primary (VX2) livertumors and lung metastases (Kim et al., 2006). As in the mouse models,1×10⁹ PFU of intravenous vvDD had significant anti-tumor effects (FIG.9A). The vvDD virus was also capable of inhibiting the outgrowth ofmicroscopic lung metastases. In order to assess additional efficacy dueto concomitant GM-CSF expression, JX-963 was compared directly to vvDD.JX-963 produced greater efficacy against the primary tumor, andcompletely blocked outgrowth of lung metastases. GM-CSF was detected inthe plasma of JX-963 treated mice by ELISA (data not shown). In additionto direct oncolytic effects, JX-963 was also found to cross-protect theanimal against the tumor by raising a CTL response against the VX2 tumorcells (FIG. 9B).

One concern in using vaccinia virus as an anti-tumor agent is that, eventhough systemic delivery to the tumor is initially possible in naïveindividuals, the immune response raised by prior exposure to the virusmay inhibit the efficacy of subsequent treatment. A strong anti-viralantibody response was raised within 3 weeks of initial infection in therabbits tested (FIG. 14). To study the feasibility of repeat dosingafter neutralizing antibody formation, four rabbits that had initiallyresponded to treatment but had tumor progression after four weeks off oftherapy were re-treated. 1×10⁹ PFU of JX-963 delivered intravenously at6 weeks after the initial treatment resulted in a decrease in primarytumor size in 3 of 4 animals treated (FIG. 9C).

Therefore, by selecting vaccinia virus, that has evolved to spreadthrough the hematopoietic system, and screening strains for tumorselective replication the inventor was able to find a virus capable ofsystemic tumor delivery with rapid oncolytic effects. In order toimprove the safety of this virus several deletions capable of increasingits therapeutic index were introduced, their mechanism of actiondescribed and their biodistribution examined in vivo. Dramatictherapeutic effects against large primary tumors following systemicdelivery were demonstrated. Finally, because it is unlikely all tumorcells will be infected, even following systemic viral delivery, GM-CSFwas expressed from this viral backbone. The addition of GM-CSF was foundto increase the effectiveness of this virus against primary tumors,prevent the outgrowth of micrometastases, and produced an anti-tumor CTLresponse. This indicates that this virus, JX-963, is capable of systemicdelivery to tumors, where it rapidly and efficiently destroys tumortissue, while sparing normal organs, and at the same time induces animmune response within the tumor that is capable of recognizing tumorantigens produced in situ. Repeat dosing was further shown to produceadditional anti-tumor effects, either by direct oncolysis or by boostingthe anti-tumor immune response. JX-963 therefore has the potential toeffectively treat a variety of tumors.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and/or methods and in the steps or in the sequence of stepsof the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents that are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

IX. REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference:

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The invention claimed is:
 1. A method of killing a cancer cell in ahuman subject comprising administering intravascularly to the subjectmultiple doses of a pharmaceutical composition comprising apharmaceutically acceptable carrier and a purified replicative vacciniavirus that: (i) lacks a functional thymidine kinase (TK) protein and(ii) expresses a nucleic acid encoding human granulocyte-macrophagecolony stimulating factor (GM-CSF), wherein said dose comprises at least1×10⁹ plaque forming units (pfu) of the virus.
 2. The method of claim 1,wherein the vaccinia virus is administered intravenously.
 3. The methodof claim 1, wherein the vaccinia virus is administered intraarterially.4. The method of claim 1, wherein the vaccinia virus is the Wyeth orWestern Reserve (WR) strain.
 5. The method of claim 4, wherein thevaccinia virus is the WR strain.
 6. The method of claim 1, wherein thesubject is administered the vaccinia virus multiple times.
 7. The methodof claim 6, Wherein a second treatment occurs within 3 weeks of a firsttreatment.
 8. The method of claim 7, wherein the second treatment occurswithin 2 weeks of the first treatment.
 9. The method of claim 6, whereinthe same dose is administered.
 10. The method of claim 1, wherein theadministration occurs intravascularly by injection, intravenous drip,bolus or pump.
 11. The method of claim 1, wherein the subject has lungcancer, colorectal cancer, breast cancer, prostate cancer, pancreaticcancer, hepatocellular cancer, leukemia, lymphoma, myeloma, melanoma,gastrointestinal cancer, renal cancer, head and neck cancer,mesothelioma, or ovarian cancer.
 12. The method of claim 1, wherein saiddose comprises 10⁹ to 10¹⁰ pfu of the virus.
 13. The method of claim 1,wherein expression of a nucleic acid encoding humangranulocyte-macrophage colony stimulating factor (GM-CSF) is directed bya synthetic vaccinia early/late promoter.
 14. A method for treating oneor more metastases in a human subject comprising administeringintravascularly to the subject multiple doses of a pharmaceuticalcomposition comprising a pharmaceutically acceptble carrier and apurified replicative vaccinia virus that: (i) lacks a functionalthymidine kinase (TK) protein and (ii) expresses a nucleic acid encodinggranulocyte-macrophage colony stimulating factor (GM-CSF), wherein saiddose comprises at least 1×10⁹ plaque forming units (pfu) of the virus.15. The method of claim 14, wherein expression of a nucleic acidencoding human granulocyte-macrophage colony stimulating factor (GM-CSF)is directed by a synthetic vaccinia early/late promoter.
 16. The methodof claim 14, wherein 10⁹ to 10¹⁰ pfu of the vaccinia virus isintravascularly administered to the subject.
 17. The method of claim 14,wherein the vaccinia virus is administered intravenously.
 18. The methodof claim 14, wherein the vaccinia virus is the Wyeth or Western Reserve(WR) strain.
 19. The method of claim 18, wherein the vaccinia virus isthe WR strain.
 20. The method of claim 1, wherein the virus lacks afunctional VGF gene.
 21. The method of claim 14, wherein the virus lacksa functional VGF gene.
 22. The method of claim 1, wherein the timeinterval between administering each consecutive dose is at least oneday.
 23. The method of claim 14, wherein the time interval betweenadministering each consecutive dose is at least one day.